Effect of Penetration Enhancers as Cosurfactants on Transdermal Delivery of Caffeine and Using Microemulsions

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript describes the formulation and characterization of caffeine microemulsion for transdermal delivery. Caffeine is a key molecule either as cosmeceutical or pharmaceutical and hence formulation attempts of caffeine is a highly sought area. References are adequate in number and up to date, and the work addresses a knowledge gap of finding novel formulations for caffeine. Only few minor comments:

1- Please provide an overview of all reported caffeine microemulsion formulations in addition to those in (lines 71-81). This is important to highlight the novelty of the work.

2- Line 96, state the molar concentration of PBS.

3- Line 106, correct agilant to agilent and line 202 and figure 2 correct disposition to deposition.

4- Line 210, state the model of the centrifuge.

5- Figure 1 and 2, the S.D. values seem very high, why didn't the authors try including more replicates for the experiment?

6- Statistics need to be revised, and a table for the values in Figures 1 and 2 need to be present in the manuscript. S.D. are drawn only as +S.D. not ±, and it seems that Me8 will not be statistically significant from control.

 

 

Author Response

1- Please provide an overview of all reported caffeine microemulsion formulations in addition to those in (lines 71-81). This is important to highlight the novelty of the work.

We thank the reviewer for this important suggestion. The Introduction has been revised to include a broader overview of previously reported caffeine microemulsion and related delivery systems in order to better contextualize the current work and highlight its novelty.

Specifically, additional studies have been incorporated discussing the role of formulation composition, carrier systems, and physicochemical factors influencing caffeine permeation. These include studies on vesicular and colloidal systems, formulation microstructure, surfactant properties, and recent advances in microemulsion-based delivery approaches.

This expanded discussion now more clearly emphasizes the existing knowledge while identifying a key gap—namely, the lack of systematic investigation of cosurfactant physicochemical properties under controlled formulation conditions—which forms the basis of the present study.

 

Previous studies have examined formulation variables, skin models, and permeation behavior of caffeine and approached the topic from different angles. For instance, Zhang et al. investigated the effect of varying water and oil ratios on the skin permeation of caffeine—a hydrophilic compound—and compared it to lidocaine, a hydrophobic molecule[15]. Sintov et al.  evaluated caffeine’s ability to penetrate the skin of various animals, including rats, rabbits, and pigs, and assessed whether using fresh versus frozen/thawed skin altered permeation results[13]. Limpongsa et al. [16] developed grapefruit oil-based microemulsions for transdermal caffeine delivery, examining the impact of formulation parameters such as grapefruit oil concentration, Tween 20, cosurfactant type and quantity, and caffeine content on both the microemulsion properties and skin permeation, using pig ear skin as the model.

In addition, Abd et al. reported that the type of vesicular or colloidal carrier system, including microemulsion-like structures, significantly influences caffeine penetration and deposition in human skin, underscoring the critical role of formulation microstructure in delivery efficiency[17]. Similarly, Todo et al. demonstrated that modifying the microenvironment of caffeine through formulation strategies can markedly enhance its skin permeation, particularly for hydrophilic compounds. Furthermore, Mekarun et al. investigated caffeine-containing emulsified systems and highlighted the importance of surfactant composition, hydrophilic–lipophilic balance (HLB), and oil phase characteristics in determining formulation stability and drug release behavior[18].

More recently, Salimi et al. reported enhanced transfollicular delivery of caffeine using optimized oil-in-water microemulsions, emphasizing the role of formulation design in targeting alternative skin penetration pathways[19]. In agreement with these findings, Bolzinger et al. demonstrated that oil-in-water microemulsions can achieve faster permeation and improved skin retention of caffeine compared to conventional emulsions and gel systems, further highlighting the advantages of microemulsion-based delivery platforms[20].

 

 

 

 

2- Line 96, state the molar concentration of PBS.

Thank you for this comment. The molar concentration of PBS has been specified as 100 mM in the revised manuscript.

3- Line 106, correct agilant to agilent and line 202 and figure 2 correct disposition to deposition.
Thank you for identifying these errors. All typographical corrections have been made in the revised manuscript.

4- Line 210, state the model of the centrifuge.
Thank you for this suggestion. The centrifuge model has been added to the manuscript as follows:

IKA centrifuge (G-L, Germany).

5- Figure 1 and 2, the S.D. values seem very high, why didn't the authors try including more replicates for the experiment?

We thank the reviewer for this important comment. The observed variability (standard deviation) in Figures 1 and 2 is primarily attributed to the inherent biological variability associated with the use of human cadaver skin, which is well recognized in transdermal permeation studies. Factors such as donor variability, differences in skin structure, and heterogeneity of the stratum corneum can contribute to increased data dispersion.

In this study, experiments were conducted using n = 5 replicates, which is consistent with standard practice in ex vivo permeation studies. Increasing the number of replicates may reduce variability; however, it is often limited by tissue availability and ethical considerations associated with human skin use.

Importantly, despite this variability, statistically significant differences were observed between certain formulations and the control. For example, ME8 showed a statistically significant increase in both epidermal and dermal caffeine deposition compared to control (p < 0.05), while other formulations showed varying degrees of significance.

We have also clarified in the revised manuscript that the primary objective of this study was to compare each formulation with the control, rather than to perform pairwise comparisons between all formulations. This approach was selected to specifically evaluate the effect of incorporating surfactants and cosurfactants relative to a baseline system (caffeine in PBS).

 

 

6- Statistics need to be revised, and a table for the values in Figures 1 and 2 needs to be present in the manuscript. S.D. are drawn only as +S.D. not ±, and it seems that Me8 will not be statistically significant from control.
We thank the reviewer for these valuable suggestions and have revised the manuscript accordingly.

The statistical analysis has been clarified to emphasize that the primary objective of this study was to compare each formulation with the control, rather than to perform pairwise comparisons between all formulations. This approach was selected to specifically evaluate the effect of incorporating surfactants and cosurfactants on caffeine permeation relative to a baseline system (caffeine in PBS).

The statistical comparisons presented in Table 4 (flux, permeability coefficient, and enhancement ratio) and in the skin deposition analysis were therefore performed against the control formulation, and not between formulations. which is consistent with the study objective.

Third, the graphical representation of variability has been corrected, and all standard deviation values are now presented as mean ± SD throughout the manuscript.

Finally, the statistical significance of ME8 compared to control has been carefully re-evaluated and confirmed using the appropriate statistical test (one-way ANOVA with Dunnett’s post hoc test), showing a significant difference (p < 0.05), as reported in the revised manuscript.
These revisions improve the clarity and interpretation of the statistical analysis.

Reviewer 2 Report

Comments and Suggestions for Authors

1. Nanoemulsion is unstable system. This us major limitation of thus system compared to SLN, NLC. Please, remove this from introduction part.
2. Please move the aim of study to last part of introduction.
3. Remove 2.2. and make UPLC 2.2. and complete with same sequence.
4. State that 25 is temperature of column during analysis.
5. Why run time 10 minutes and drug elated at 4 minutes.
6. How authors measure solubility based on volumetric. These are viscous solvents. How they ensure taking same volume which could stick to withdrawing agent.
7. The prepared formulations are not clearly described in method section. Please revise to make sure it clear for readers. 
8. Why authors present solubility in phosphate buffer. Also, categories surfactant and cosurfactant in Table. 
9. Please make statistical analysis for data and consider refer to significant while choose components.
10. Result section needs to be restructured. Please make logical flow of presented data. Pseudo diagram usually performed first to select optimized formulation.
11. Please avoid using long paragraph to present data. It is already presented in Table and figure. Reduce the length of unnecessary sections. No need to mention properties and structure of surfactant and cosurfactant.
12. Merge table 5 and 6. Also Merge table 7 and 8.

Author Response

Comment: Nanoemulsion is unstable system. This us major limitation of thus system compared to SLN, NLC. Please, remove this from introduction part.

Thank you for your comment. Our manuscript does not include a discussion of nanoemulsions in the Introduction. The study is focused exclusively on microemulsion systems, which are thermodynamically stable formulations, as correctly described in the manuscript. Therefore, no statement regarding nanoemulsion instability is present in the current version.

Comment: Please move the aim of study to last part of introduction.
We thank the reviewer for this suggestion. The aim of the study has been moved to the final paragraph of the Introduction.

 

Comment: Remove 2.2. and make UPLC 2.2. and complete with same sequence.
We thank the reviewer for this suggestion. The section numbering has been revised accordingly. The UPLC section has been updated to Section 2.2, and all subsequent sections have been renumbered to maintain a consistent sequence throughout the Methods section.

Comment: State that 25 is the temperature of the column during analysis.
We thank the reviewer for this suggestion.
Agilent ChemStation software (OpenLab CDS, ChemStation Edition, Rev. C.01.10, Agilent Technologies, Santa Clara, CA, USA) was used for data acquisition and analysis. An Agilent C18 column (150 mm × 4.6 mm, 5.0 µm particle size) was used as the stationary phase, and the column temperature was maintained at 25 °C during analysis.

Comment: Why run time 10 minutes and drug elated at 4 minutes.

We thank the reviewer for this comment. Although caffeine elutes at approximately 4 minutes, the total run time was set to 10 minutes to ensure adequate column re-equilibration and to allow detection of any potential late-eluting impurities or degradation products. This extended run time enhances the robustness, reliability, and reproducibility of the analytical method.

 

Comment: How authors measure solubility based on volumetric. These are viscous solvents. How they ensure taking the same volume which could stick to withdrawing agent.
We thank the reviewer for this important comment. The solubility measurements were performed using a gravimetric (weight-based) method, rather than a volumetric approach. Specifically, all solvents were weighed using an analytical balance, and excess caffeine was added to achieve saturation. This approach avoids inaccuracies associated with volumetric measurements of viscous solvents, such as adherence to pipette surfaces or incomplete transfer. By using weight-based measurements, we ensured consistency and accuracy across all samples regardless of solvent viscosity.

 

Comment: The prepared formulations are not clearly described in method section. Please revise to make sure it clear for readers.
We thank the reviewer for this helpful comment. The Methods section has been revised to clarify the selection process of the formulations. Specifically, it is now stated that drug-loaded microemulsions were prepared based on compositions identified from the pseudoternary phase diagrams, and only formulations within the microemulsion region (clear and isotropic systems) were selected for further evaluation. This clarification improves the transparency and reproducibility of the formulation strategy.

Comment: Why authors present solubility in phosphate buffer. Also, categories surfactant and cosurfactant in Table.
The aim of this paper was to study the behavior of these surfactants and co-surfactants, PBS for sink condition/ the highest solubility for caffeine 

Comment: Please make statistical analysis for data and consider refer to significant while choose components.

We thank the reviewer for this valuable suggestion. Statistical analysis has been performed and clarified throughout the revised manuscript. Specifically, data were analyzed using one-way ANOVA followed by Dunnett’s post hoc test, with each formulation compared to the control (caffeine in PBS).

The selection and evaluation of formulation components were based on both the magnitude of enhancement and statistical significance relative to the control. It is important to note that the primary objective of this study was not to perform pairwise statistical optimization between all formulations, but rather to evaluate the effect of incorporating different cosurfactants relative to a baseline system.

These clarifications have been incorporated into the revised manuscript.

 

 

Comment: Result section needs to be restructured. Please make logical flow of presented data. Pseudo diagram usually performed first to select optimized formulation.
We thank the reviewer for this valuable suggestion. The Results and Discussion section has been carefully restructured to improve the logical flow and clarity of data presentation.

Specifically, the sequence has been revised to reflect the experimental workflow, beginning with the pseudoternary phase diagram analysis to identify the microemulsion region and guide formulation selection. This is followed by solubility studies, permeation results, and skin deposition analysis.

Comment: Please avoid using long paragraph to present data. It is already presented in Table and figure. Reduce the length of unnecessary sections. No need to mention properties and structure of surfactant and cosurfactant.

We thank the reviewer for this valuable suggestion. The Results and Discussion section has been revised to improve clarity and conciseness.

Comment: Merge table 5 and 6. Also Merge table 7 and 8.

We thank the reviewer for this helpful suggestion. The tables have been reorganized to improve clarity and reduce redundancy.

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have explored the impact of a series of co-surfactants in the preparation of microemulsions for topical drug delivery, and compared the benefit of the co-surfactants on the in vitro skin permeability of a model hydrophilic compound, caffeine.  The  study is appropriately designed and executed. A few point ought to be addressed to improve the suitability of the  manuscript for consideration publication in the journal, as follows:

Page 1, line 17- 18: it is preferable to describe the co-surfactants by their compendial names or accepted trivial names rather than by brand names that link them to a specific commercial supplier that has been involved in the preparation of the manuscript. So in the abstract we should just have polysorbate 80 (PS80), polyoxyl 40 castor oil (RH40), polysorbate 20 (PS20) and octadodecanol (OD).

Page 2, line 60-61: is caffeine used in this work simply as a pharmaceutically relevant, hydrophilic, small molecule model compound, with relatively straightforward analytical approaches?  This work is not around  formulating it for improving bioavailability transdermally, with a future clinical study in mind that these lines might suggest? 

Pages 2 -3, lines 90-95.  Please use compendial or accepted trivial names for excipients used, with brand and supplier in brackets - polysorbate 80 (PS80, Kolliphor® PS 80, BASF, Tarrytown, NY, USA), polysorbate 20 (PS20, Kolliphor® PS 20, BASF, Tarrytown, NY, USA), caprylocaproyl polyoxyl-8-glycerides, Labrasol®, Gattefosse, Parasmus, NJ, USA), etc.

Page 4, line 148:  authors state caffeine was full dissolved in the oil/surfactant/co-surfactant mix. Table 3 suggests maximum solubility in the most favourable component of these components is about 3mg yet caffeine concentration in final microemulsion is 4mg.mL.  Was the caffeine really fully dissolved in the oil/surfactant/co-surfactant phase?

Page 4, line 153, section is numbered 2.2.6  but prior section was 2.2.3. On page 5 and 6, sections following are numbered 2.2.4 and 2.2.5, then after 2.2.5 the next section on page 6 is 2.2.7. Sections may be have copied and pasted from a draft out of order? Please correct section numbers.

Page 6 lines 225-244: please stick to the previously given code for the ingredients, PS 20, PS 80 , OD (as per line 250 on page 7) and avoid using brand names.

Pages 11 - 12: as above comment, excipient  brand names occur in several places on these pages, please amend.

Author Response

Comment: Page 1, line 17- 18: it is preferable to describe the co-surfactants by their compendial names or accepted trivial names rather than by brand names that link them to a specific commercial supplier that has been involved in the preparation of the manuscript. So in the abstract we should just have polysorbate 80 (PS80), polyoxyl 40 castor oil (RH40), polysorbate 20 (PS20) and octadodecanol (OD).

We thank the reviewer for this helpful suggestion,this issue has been resolved in teh updated manuscript

Comment:Page 2, line 60-61: is caffeine used in this work simply as a pharmaceutically relevant, hydrophilic, small molecule model compound, with relatively straightforward analytical approaches?  This work is not around  formulating it for improving bioavailability transdermally, with a future clinical study in mind that these lines might suggest? 

Comment:Pages 2 -3, lines 90-95.  Please use compendial or accepted trivial names for excipients used, with brand and supplier in brackets - polysorbate 80 (PS80, Kolliphor® PS 80, BASF, Tarrytown, NY, USA), polysorbate 20 (PS20, Kolliphor® PS 20, BASF, Tarrytown, NY, USA), caprylocaproyl polyoxyl-8-glycerides, Labrasol®, Gattefosse, Parasmus, NJ, USA), etc.

We thank the reviewer for this helpful suggestion,this issue has been resolved in teh updated manuscript

Comment:Page 4, line 148:  authors state caffeine was full dissolved in the oil/surfactant/co-surfactant mix. Table 3 suggests maximum solubility in the most favourable component of these components is about 3mg yet caffeine concentration in final microemulsion is 4mg.mL.  Was the caffeine really fully dissolved in the oil/surfactant/co-surfactant phase?

We thank the reviewer for this important observation. Although Table 3 reports the solubility of caffeine in individual components, the drug-loaded microemulsions consist of a combined system of oil, surfactant, cosurfactant, and water, which significantly enhances the overall solubilization capacity.

In such multicomponent systems, solubility is not limited by the individual solubility in each component but is governed by the collective solubilizing environment, including interfacial regions and mixed micellar structures.

In our study, caffeine was visually observed to be completely dissolved in the oil/surfactant/cosurfactant mixture prior to water addition, with no signs of precipitation or turbidity upon preparation or during subsequent evaluation. This indicates that the final formulation provided sufficient solubilization capacity to accommodate the drug concentration used (0.4% w/w).

 

Comment:Page 4, line 153, section is numbered 2.2.6  but prior section was 2.2.3. On page 5 and 6, sections following are numbered 2.2.4 and 2.2.5, then after 2.2.5 the next section on page 6 is 2.2.7. Sections may be have copied and pasted from a draft out of order?

We thank the reviewer for identifying this issue. The section numbering has been carefully reviewed and corrected throughout the Methods section to ensure a consistent and logical sequence.

Comment:Page 6 lines 225-244: please stick to the previously given code for the ingredients, PS 20, PS 80 , OD (as per line 250 on page 7) and avoid using brand names.

We thank the reviewer for this helpful suggestion,this issue has been resolved in teh updated manuscript

 

Comment:Pages 11 - 12: as above comment, excipient  brand names occur in several places on these pages, please amend. 

 

We thank the reviewer for this helpful suggestion,this issue has been resolved in teh updated manuscript

Reviewer 4 Report

Comments and Suggestions for Authors

The current study is interesting however the following should be addressed:

1-detailed methadology should be reported in the abstract (time intervals for sampling in permeation study).

2-The Zeta potential values of different formulae are not reported, these values are essential to assess colloidal stability of different formulae.

3-No morphology assessment using TEM.

4-The authors stated that the formulae were stable over 4 month and 10 month period in the result and discussion section, however the exact conditions of storage  (RH%) were not reported in the methodology section.

5-the authors stated that caffeine exhibited hydrophilic properties without reporting its log P.

5-the authors discussed the role of each ingredients within formulae on caffeine permeation, this section is better to be summerized within one paragraph as all of these are interperation of same experiment which is ex-vivo permeation section.

6-what statistical test were performed to compare values in stability section ( before and after storage), please mention test and p values.

7- the authors report pH, without discussing is these values applicable for topical application or not, also droplet size without discussing impact of Co surfactant on its value or impact of these values on permeation or deposition study.

8- revise title of table 8, remove freshly.

 

Author Response

1-detailed methadology should be reported in the abstract (time intervals for sampling in permeation study).

Thank you for your comment. The detailed methodology, including the sampling time intervals for the permeation study, is already reported in the abstract (Lines 22–24): “Receptor compartment samples were collected at 2, 4, 6, 8, 10, 12, and 24 hours, and CF permeation was quantified using a validated HPLC method.”

2-The Zeta potential values of different formulae are not reported, these values are essential to assess colloidal stability of different formulae.

Thank you for your comment. While zeta potential is important for assessing the stability of colloidal dispersions, microemulsions are thermodynamically stable systems and do not rely on surface charge for stability. In our study, stability was confirmed by the absence of phase separation and consistent physicochemical properties. Therefore, zeta potential measurement was not considered essential. This has been clarified in the revised manuscript.

3-No morphology assessment using TEM.

Thank you for your valuable comment. We agree that morphological characterization using TEM can provide additional insight into the system. However, this analysis was beyond the scope of the current study. This limitation has now been acknowledged in the revised manuscript, and we have suggested TEM analysis as part of future work to further confirm the morphology of the developed microemulsions.

4-The authors stated that the formulae were stable over 4 month and 10 month period in the result and discussion section, however the exact conditions of storage  (RH%) were not reported in the methodology section.

We thank the reviewer for this important comment. The storage conditions have now been added to the Methods section. All formulations were stored at room temperature under controlled relative humidity (53% RH) throughout the stability study. This humidity level falls within standard laboratory conditions and is appropriate for maintaining formulation consistency.

5-the authors stated that caffeine exhibited hydrophilic properties without reporting its log P.

We thank the reviewer for this comment. The log P value of caffeine (log P = 0.07) has now been added to the manuscript

5-the authors discussed the role of each ingredients within formulae on caffeine permeation, this section is better to be summerized within one paragraph as all of these are interperation of same experiment which is ex-vivo permeation section.

We thank the reviewer for this helpful suggestion. The section has been revised and consolidated into a single paragraph to provide a more concise and unified interpretation of the ex vivo permeation results, while reducing redundancy.

 

 

6-what statistical test were performed to compare values in the stability section ( before and after storage)? Please mention test and p-values.
We thank the reviewer for this important comment. Statistical analysis was performed to compare the physicochemical properties of formulations before and after storage using a paired t-test.

The results showed no statistically significant differences (p > 0.05) in droplet size, PDI, pH, viscosity, or refractive index between freshly prepared and stored formulations, confirming the stability of the systems over time.

.

 

7- the authors report pH, without discussing is these values are applicable for topical application or not, also droplet size without discussing impact of Co surfactant on its value or impact of these values on permeation or deposition study.
We thank the reviewer for this important comment. Additional discussion has been incorporated into the revised manuscript to address the relevance of pH and droplet size.

The pH values of the formulations (approximately 3.5–5.7) fall within or close to the physiological skin pH range (typically 4.5–6.5), indicating their suitability for topical application.

Furthermore, the droplet sizes of the microemulsions (ranging from ~11 to 42 nm) are within the expected nanoscale range for microemulsion systems, which is known to enhance drug permeation by increasing surface area and facilitating interaction with the stratum corneum.

The variation in droplet size observed between formulations is attributed to the different physicochemical properties of the cosurfactants, which influence interfacial tension and microemulsion structure. These differences may contribute to the observed variations in caffeine permeation and skin deposition.

These points have been added to the revised manuscript to improve interpretation of the results.

 

8- revise title of table 8, remove freshly.
We thank the reviewer for this suggestion. The table has been revised accordingly.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Thank authors for their responses. However, I cannot recommend manuscript for publication. Generally, the selected excipients for optimized formulation not performed systematically. Moreover, there are inconsistencies in flow throughout the manuscript. Kindly find below some recommendations during future work. 

1. Please avoid usage of long sentences. For example, first and last sentences in abstract. This only example. Please revise all manuscript.
2. Remove - in middle ot some words. Like en-hancer.
3. Please rewrite abstract to include main results. Not only for permeability study. Overall the conclusion section of abstract need to be reduced. 
4. In manuscript authors used microemulsion term. However, in Introduction they refer to particles less than 100nm. Is this nanoemulsion.
5. Introduction needs to be rewritten again. Three main areas need to be covered. First advantage and demand for transdermal delivery. Second the used drug delivery system. Third model drug. The literature review for each area needs to be added in its relevant area. Last paragraph aim and objectives to achieve it. So kindly move lines 64 -  60 in last paragraph. 
6. What is the rational to include IPM in Drug loaded formulation. LB high solubility. OD high permeability. What is rational for IPM. What is the benifit of perform solubility study, then select components based on literature.
7. Please reduce the length of results sectio. Be brief to avoid readers boring. All data area present in Table af figures.
8. Authors select excipients then perform pseudoternaty diagram for emulsification.

Author Response

1. Please avoid usage of long sentences. For example, first and last sentences in abstract. This only example. Please revise all manuscript.
   
   We thank the reviewer for this valuable suggestion. The manuscript has been carefully revised throughout to improve readability and clarity. Long and complex sentences have been shortened and simplified, particularly in the Abstract, Introduction, and Results sections.
   
   
   
2. Remove - in middle ot some words. Like en-hancer.
   
   We thank the reviewer for identifying this issue. Hyphenation errors introduced during formatting have been corrected throughout the manuscript.
   
   
   
3. Please rewrite abstract to include main results. Not only for permeability study. Overall the conclusion section of abstract need to be reduced.
   
   We thank the reviewer for this suggestion. The Abstract has been revised to include the main experimental findings, including solubilty, permeation, and stability.

 

1. In manuscript authors used microemulsion term. However, in Introduction they refer to particles less than 100nm. Is this nanoemulsion.
   
   We thank the reviewer for this important comment. Microemulsion exhibited droplet sizes below 100 nm. While microemulsions and nanoemulsions may overlap in droplet size range, nanoemulsions are kinetically stable systems that generally require external energy input for formation, whereas the formulations in this study formed spontaneously and demonstrated characteristics consistent with microemulsion systems.
   As reported by Lawrence et al., Microemulsion typically in the range 60–200 nm.

Lawrence MJ, Rees GD. Microemulsion-based media as novel drug delivery systems. Adv Drug Deliv Rev. 2000;45(1):89–121.

1. Introduction needs to be rewritten again. Three main areas need to be covered. First advantage and demand for transdermal delivery. Second the used drug delivery system. Third model drug. The literature review for each area needs to be added in its relevant area. Last paragraph aim and objectives to achieve it. So kindly move lines 64 -  60 in last paragraph.
   We thank the reviewer for this valuable suggestion. The Introduction was already structured to address the advantages and demand for transdermal drug delivery, the characteristics of microemulsion systems, and caffeine as the model drug, with the relevant literature incorporated within each section.
   
   
   
2. What is the rational to include IPM in Drug loaded formulation. LB high solubility. OD high permeability. What is rational for IPM. What is the benefit of performing solubility study, then select components based on literature.

We thank the reviewer for this important comment. IPM was selected as the oil phase based on both literature evidence and the experimental findings obtained from the solubility study.

IPM is widely used in topical and transdermal formulations due to its favorable safety profile, excellent solubilization capacity, and well-documented penetration-enhancing properties. It is known to interact with the lipid domains of the stratum corneum, increasing lipid fluidity and reducing barrier resistance, thereby facilitating drug diffusion through the skin. In addition, IPM contributes to the formation of stable microemulsion systems with nanoscale droplet distribution.

Although literature reports were considered during excipient selection, the solubility study was still necessary to experimentally confirm the compatibility and solubilization behavior of caffeine within the selected formulation components under the conditions used in this study. Solubility screening is an essential step in formulation development, as it supports appropriate component selection, drug loading capacity, formulation stability, and reproducibility.

In this study, Labrasol (LS) and IPM were selected as the surfactant and oil phase, respectively. Both components can also function as cosolvents and exhibit strong solubilization capacity for hydrophilic and lipophilic compounds. Furthermore, they are recognized for their safety and their ability to enhance skin permeation [51].

 

 

1. Please reduce the length of results section. Be brief to avoid readers boring. All data area present in Table af figures.
   
   We thank the reviewer for this suggestion. The Results and Discussion section has been significantly condensed to reduce repetition and improve readability
   
   
2. Authors select excipients then perform pseudoternaty diagram for emulsification.

We thank the reviewer for this comment. The manuscript has been revised to clarify the workflow for formulation development. Excipients were initially selected based on their physicochemical properties, safety profile, solubilization capacity, and reported transdermal enhancement potential. Subsequently, pseudoternary phase diagrams were constructed to identify the microemulsion region and select suitable formulations for further evaluation.

Reviewer 4 Report

Comments and Suggestions for Authors

The uploaded  manuscript version doesn't include any of the requested corrections, please revise. 

Regarding the authors report, all the information required are answered, however the first comment was missing the word not  ( the abstract section should not include detailed methodology regarding sampling intervals ), please revise and upload the revised version.

Author Response

Regarding the authors report, all the information required are answered, however the first comment was missing the word not  ( the abstract section should not include detailed methodology regarding sampling intervals ), please revise and upload the revised version.

We thank the reviewer for this valuable comment.The abstract has been updated

Round 3

Reviewer 2 Report

Comments and Suggestions for Authors

Thank the authors for addressing comments. However, there is important issue need to be addressed before publication.

Why authors include IPM as excipient in Drug loaded formulation even with low drug solubility. Authors needs to classify drug solubility based on its composition rule (surfactant. Cosurfactant,...). Remove solubility in phosphate buffer. No need for such information.

Author Response

We thank the reviewer for this important comment. We agree that caffeine showed low solubility in IPM when tested as an individual component. However, IPM was not selected as the primary solubilizing excipient for caffeine. Rather, IPM was included as the oil phase of the microemulsion system because of its well-established role in topical and transdermal formulations as a skin penetration enhancer and oil-phase component.

Although caffeine is hydrophilic and has limited solubility in IPM alone, the final microemulsion is a multicomponent system composed of oil, surfactant, cosurfactant, and water. Therefore, caffeine solubilization in the final formulation is governed by the combined solubilizing capacity of the full microemulsion system rather than by its solubility in IPM alone. In this system, Labrasol and the selected cosurfactants contribute substantially to drug solubilization, while IPM contributes to microemulsion formation, nanoscale droplet distribution, and enhanced skin permeation.

In response to the reviewer’s suggestion, the solubility data have been reorganized according to the functional role of each excipient in the formulation, including oil phase, surfactant, and cosurfactant. The phosphate buffer solubility data have been deleted from the solubility section to maintain the focus on excipient selection. 

Reviewer 4 Report

Comments and Suggestions for Authors

The manuscript can be accepted in its current form.

Author Response

We thank the reviewer for this important comment. We agree that caffeine showed low solubility in IPM when tested as an individual component. However, IPM was not selected as the primary solubilizing excipient for caffeine. Rather, IPM was included as the oil phase of the microemulsion system because of its well-established role in topical and transdermal formulations as a skin penetration enhancer and oil-phase component.

Although caffeine is hydrophilic and has limited solubility in IPM alone, the final microemulsion is a multicomponent system composed of oil, surfactant, cosurfactant, and water. Therefore, caffeine solubilization in the final formulation is governed by the combined solubilizing capacity of the full microemulsion system rather than by its solubility in IPM alone. 

In response to the reviewer’s suggestion, the solubility data have been reorganized according to the functional role of each excipient in the formulation, including oil phase, surfactant, and cosurfactant. The phosphate buffer solubility data have been deleted from the solubility section to maintain the focus on excipient selection (Table 3).

Round 4

Reviewer 2 Report

Comments and Suggestions for Authors

Manuscript can be published now.