Topical Biocompatible Fluconazole-Loaded Microemulsions Based on Essential Oils and Sucrose Esters: Formulation Design Based on Pseudo-Ternary Phase Diagrams and Physicochemical Characterization

Round 1

Reviewer 1 Report

This manuscript evaluates the effect of hydrocarbon chain length of sucrose ester and its concentration, and of the essential oil type and its concentration on the phase behavior. In my opinion, the article does not represent a novelty in the field of study neither a significative relevance. The methodology used to obtain the data is not suitable to provide evidence for the conclusion of the manuscript. In this context, I do not support the publication of the manuscript.

Some considerations to improve the article:

• The introduction of the manuscript describes that solubility studies of fluconazole and sucrose esters in essential and synthetic oils have also been performed however, there is no detailed information on its relevance. I strongly recommend the authors to rewrite the introduction considering the comment above.
• Table 3. Apparent solubility of fluconazole in the selected essential oils shows slight differences in the weight measurement (Ex: Lavender essential oil 7.85±0.25 indicating differences of 0.05 mg. The authors should describe accurately how they perform these measurements.
• The authors present differences between fluconazole-loaded microemulsions formulation, revealing differences below 10nm on particle diameter. These values were obtained through DLS measurements. It is well known that size measurements provided by DLS are not very reliable under 50nm. It is hard to believe that size differences below 10nm are led by modification of the formulations. In my opinion if the intention of the authors is to demonstrate such low variations in size distribution, they must re-analyse the samples using an appropriate technique such NTA.

 

Author Response

Comment 1:

The introduction of the manuscript describes that solubility studies of fluconazole and sucrose esters in essential and synthetic oils have also been performed however, there is no detailed information on its relevance. I strongly recommend the authors to rewrite the introduction considering the comment above.

 

Answer:

As it was mentioned in the last paragraph of the "Introduction" section, this manuscript describes the first step, namely the formulation phase of development of some topical biocompatible FZ-loaded microemulsions based on essential oils and sucrose esters. In this formulation study, a generally sound methodology was applied, described and discussed and therefore, we considered that is no need to highlight in the "Introduction" section the importance of solubility studies for the formulation stage of microemulsions as a pharmaceutical dosage form. However, in accomplishing the reviewer's recommendation, we have completed the "Introduction" section as follows:

"Moreover, solubility studies of fluconazole and sucrose esters in essential and synthetic oils have also been performed, having in mind the following generally accepted strategies of MEs formulation: a) currently, the formulation study of a poorly water-soluble drug as ME is preceded by experimental determination of its solubility in various oils to select the most powerful oily solubilizer as lipophilic phase of the ME [13,14,17,27]; b) when a solid surfactant is selected as ME stabilizer, determination of its solubility in the lipophilic components is required, because this property significantly affect the phase behavior and the size of the microemulsion area in the ternary oil-surfactant/cosurfactant-water systems [13, 20]. Consequently, the study of drug and solid surfactant solubility in lipophilic components represent the starting point to address the formulation of stable, safe microemulsion vehicles with minimum content of surfactant and cosurfactant."     

 

Comment 2:

Table 3. Apparent solubility of fluconazole in the selected essential oils shows slight differences in the weight measurement (Ex: Lavender essential oil 7.85±0.25 indicating differences of 0.05 mg. The authors should describe accurately how they perform these measurements.

 

Answer:

Please note that we have completed the description of the method for determining the apparent solubility of fluconazole in essential oils:

"Briefly, an accurately weighted amount (Kern ABJ-NM/ABS-N analytical balance, Kern&Sohn GmbH, Germany) of fluconazole (1 mg) was added to 1 mL of essential oil, then the systems were sonicated for 15 min to accelerate the drug dissolution and the FZ solubility was visually evaluated after 24 h. For the essential oils, in which the entire added amount of FZ was dissolved, the technique described above was repeated consecutively until a precipitate appeared. The apparent solubility was expressed as total amount of drug (in mg) added in 1 mL essential oil until saturation occurred."

 

 

Comment 3:

The authors present differences between fluconazole-loaded microemulsions formulation, revealing differences below 10 nm on particle diameter. These values were obtained through DLS measurements. It is well known that size measurements provided by DLS are not very reliable under 50 nm. It is hard to believe that size differences below 10 nm are led by modification of the formulations. In my opinion if the intention of the authors is to demonstrate such low variations in size distribution, they must re-analyse the samples using an appropriate technique such NTA.

 

Answer:

A careful survey of the manuscript reveals that we do not intended to determine the polydispersity of the nanoparticle populations in the microemulsions, as is the case of NTA. Instead, we aimed to obtain microemulsions with nanoparticles below 20 nm, suitable for topical application.

 

Moreover, for the reasons below, we do not agree with the statement: ‘DLS measurements are not very reliable under 50 nm. It is hard to believe that …..….such NTA.’

First, our microemulsions have droplet sizes in-between 10 and 20 nm (See Table 7 of the Manuscript), which are far below of 50 nm.

Second, we did the DLS measurements on a Malvern Zetasizer Nano ZS instrument having a measuring scale from 0.3 to 10,000 nm [1]. This is sustained by a technical note from the producer showing the capability of the apparatus to accurately measure a wide peak at 0.6 nm in a concentrated solution of sucrose in water with a minimum detectable particle size of 0.3nm [2]. On the other hand, the NTA instruments have measuring scale from 10 to 1,000 nm [3]. In addition, the minimum detectable size (MDS) of NTA depends on the refractive index of particles. MDS can be around 9-15 nm, but only for materials of a high refractive index like colloidal silver [4]. Obviously, this case is completely different of ours, and allows concluding that NTA is not suitable for our microemulsions.

 

References

1. https://www.chem.uci.edu/~dmitryf/manuals/Malvern%20Zetasizer%20ZS%20DLS%20user%20manual.pdf.
2. Malvern Technical note KB002657 – MRK1535-01.
3. https://www.malvernpanalytical.com/en/products/technology/light-scattering/nanoparticle-tracking-analysis).
4. Bob Carr and Andrew Malloy, NanoParticle Tracking Analysis – The NANOSIGHT system, www.nanosight.co.uk.

 

Reviewer 2 Report

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The authors provide a pharmaceutical development of microemulsions based in essential oils combined with sucrose esters. As a result, the importance of the hydrocarbon chain length of both components is pointed out. The study is interesting, and it is well designed and carried out. Just one modification and a few considerations are proposed:

Modification:

1. In table 6 refer to the denomination D1616 or D1216 since it is how they are referred to in the text.

Considerations:

1. it is taken for granted that they are biocompatible without testing it, justified in the literature. nothing is said about compatibility between components. It would be good in future studies to verify this point.
2. Line 85 refers to “the hypothesis that they could potentiate through synergism the antifungal effect of FZ”. This hypothesis is not proven. It would be interesting to study this in the future.
3. In further studies, the objective could be expanded and study whether this combination of EO and SE achieves better systems and with greater penetration capacity than without combining them.

 

Author Response

Comment 1:

In table 6 refer to the denomination D1616 or D1216 since it is how they are referred to in the text.

Answer:

Please note that we have used denomination D1616 and D1216 in table 6:

 

Oil phase component

Amount of D1216 (mg) dissolved in 1 mL oil

Apparent solubility of D1216

Amount of D1616 (mg) dissolved in 1 mL oil

Apparent solubility of D1616

 

Consideration 1:

It is taken for granted that they are biocompatible without testing it, justified in the literature. nothing is said about compatibility between components. It would be good in future studies to verify this point.

Answer:

We considered that the components of the studied fluconazole-loaded microemulsion formulations are compatible, because during the whole period of observation and storage, no changes of organoleptic characters (appearance, color, odor, homogeneity) were observed.

 

Consideration 2:

Line 85 refers to “the hypothesis that they could potentiate through synergism the antifungal effect of FZ”. This hypothesis is not proven. It would be interesting to study this in the future.

Answer:

The above mentioned hypothesis was also studied and proved by our research group, and will be reported in another work.

Round 2

Reviewer 1 Report

After the first revision of the manuscript the authors do not provided enough evidence to remove my concerns. I think that methodology used to evaluate the nanoparticle size is inadequate and therefore a large part of the article should be reconsidered. The authors provide Malvern technical notes as a supporting bibliography that measurements in the range of 0.3 to 10.000 nm are correct. Beyond the material provided by the manufacturer, there is no scientific literature on the determination of nanometric sizes in that range, and even less on changes below 10nm, using this methodology.

 

In this context I do not consider supporting this manuscript for its publication.

 

Author Response

We answered and did provided a documented response to Reviewer’s #1, Comment #3 of the Revision #1.

Until today the reviewer did not bring any proof that our methodology is inadequate. We notice that we brought all the necessary info to prove that this method is adequate to measure nanoparticles, by quoting references from producer, catalogues, etc.

We notice that the burden of proof, to demonstrate that our method is inadequate, is on reviewer’s side.