In Vitro Antioxidant Activity and In Vivo Topical Efficacy of Lipid Nanoparticles Co-Loading Idebenone and Tocopheryl Acetate

Round 1

Reviewer 1 Report

While lipid nanoparticle as drug delivery agent is a topic of immense interest to the scientific community, loading IDE on NLC is not a novel step as reflected in the authors own words (line 338) about reference 43. The title focuses on the co-loading of IDE/VitE and their synergic effect but Figure 6 & 7 as well as the conclusions emphasize the positive roles of VitE only. Overall the manuscript needs to be written more precisely, stating what improvement is intended over currently existing knowledge and has this been achieved.

# ‘good technological properties’ does not sound appropriate in line 29. Needs to be rephrased.

# In Figure 1 , distribution plots for particle size should be included from the TEM experiments.

#Figure 3 and Figure 4 are very unclear and need to be explained better. The use of codes a,b,c,d in these figures is completely incomprehensible and must be simplified.

# From the results and discussion section IDE loaded SLN seems to have much better reducing power and radical scavenging ability unlike what is stated in the conclusion.

# Line 322 says that using IDE did not increase the hydration ability of either SNL or NLC. Is this not contradictory to what is envisioned?

# Authors are providing too many references. Only the most relevant ones need to be included. Also, instead of frequently referring to what has been done by the authors in the past studies, it is more important to precisely focus on the current study.

Author Response

Reviewer 1

 

While lipid nanoparticle as drug delivery agent is a topic of immense interest to the scientific community, loading IDE on NLC is not a novel step as reflected in the authors own words (line 338) about reference 43. The title focuses on the co-loading of IDE/VitE and their synergic effect but Figure 6 & 7 as well as the conclusions emphasize the positive roles of VitE only. Overall the manuscript needs to be written more precisely, stating what improvement is intended over currently existing knowledge and has this been achieved.

Answer

The aim of this work is clearly defined in the introduction (line 111). We designed novel nanostructured lipid carriers (NLC) co-loading two antioxidants to obtain a synergic effect. As reported at line 103, to date, a variety of oils without any intrinsic pharmacological activity has been investigated to obtain NLC. In this work, we used an oil (vitamin E acetate) with antioxidant activity to produce NLC to obtain a synergic effect with the antioxidant activity of the active ingredient (Idebenone, IDE) loaded into the nanoparticles. Fig. 7 (Fig. 8 in the revised version) clearly highlights that unloaded NLC (i.e. lipid nanocarriers containing only vitamin E acetate) were less effective than IDE loaded NLC (loading both IDE and vitamin E acetate) in reducing skin pigmentation. These results highlight that co-loading IDE and vitamin E acetate resulted in a greater efficacy than loading only IDE or only vitamin E acetate. In addition, in the conclusion we pointed out the synergic effect of IDE and vitamin E acetate in reducing skin pigmentation. Therefore, it is unclear why the reviewer asserts that “the conclusions emphasize the positive roles of VitE only” and that we should state, “What improvement is intended over currently existing knowledge and has this been achieved”.

 

# ‘good technological properties’ does not sound appropriate in line 29. Needs to be rephrased.

Answer

It is unclear why “good technological properties” does not sound appropriate. We obtained lipid nanoparticles showing small particle sizes, low polydispersity indices and good stability. As all these characteristics are desirable for nanocarriers, we could sum up the properties of these lipid nanoparticles reporting that they showed good technological properties. However, to provide the readers with the opportunity to evaluate on their own the properties of these nanocarriers, we inserted the following sentence (line 30 revised version): The resulting NLC showed small particle sizes (24-42 nm), low polydispersity indices (<0.300), good stability…….

 

# In Figure 1, distribution plots for particle size should be included from the TEM experiments.

Answer

We obtained TEM images from the same samples that were analyzed by dynamic light scattering (DLS). DLS analyses showed that these samples had small particle sizes and polydispersity index values lower than 0.300, thus indicating that the distribution consisted of a single size mode. TEM images supported the homogenous size distribution of these samples. Therefore, we did not add distribution plots of these samples to avoid boring the reader with a repetition of data that can be easily read into the values obtained by DLS (reported in Table 2) and into TEM images. In addition, we have not read manuscripts published in prestigious scientific journals where the authors report distribution plots of nanoparticles samples whose polydispersity index values were low and TEM images showed nanoparticles with homogeneous sizes.

 

#Figure 3 and Figure 4 are very unclear and need to be explained better. The use of codes a,b,c,d in these figures is completely incomprehensible and must be simplified.

Answer

According to the reviewer’s suggestion, we modified the legends of Fig. 3 and 4 providing an explanation of the meaning of the letters used to report the statistical significance of the data.

In Fig. 3 we reported: ….The letters b,c,d indicate a significant difference (p<0.05), between samples loaded with different concentrations of IDE and the samples unloaded NLC and SLN (indicated with a).

In Fig.4, the representation of the statistics was simplified using a new figure and the legend was updated as follow: …..The letters b, c, indicate, for each concentration, a significant differences (p < 0.05.) between the samples loaded with different concentrations of idebenone (IDE) and the samples unloaded SLN and NLC (indicated with a).

 

# From the results and discussion section IDE loaded SLN seems to have much better reducing power and radical scavenging ability unlike what is stated in the conclusion.

Answer

In this work, we evaluated in vitro antioxidant activity (DPPH and reduction power) and cytotoxic effects on HS68 fibroblast cells (MTT test) of different concentrations (10-100 mg/ml) of NLC and SLN loading different percentages of IDE (1.0 and 1.5 % w/w). The MTT test revealed that the highest non-toxic concentration of these IDE loaded nanocarriers was 25 mg/ml. Subsequent photo-protective effects were investigated using the highest non-toxic concentration of these IDE loaded nanocarriers, as well. At this concentration, the antioxidant activity and photo-protective effect of IDE loaded SLN was similar to that of IDE loaded NLC. Therefore, in the conclusion section, we compared the antioxidant activity of IDE loaded SLN and NLC at the highest non-toxic concentration. To make clear that the comparison refers to the highest non-toxic concentration of IDE loaded lipid nanoparticles we amended the text as follows (line 592 revised version):

In vitro antioxidant assays (DPPH and reduction power) showed that, at the highest non-toxic concentration determined by the MTT test, the antioxidant activity of NLC containing different percentages of IDE (1.0 and 1.5 % w/w) was similar to that of SLN loading the same percentages of IDE. Analogously, similar photo-protective effects of IDE loaded NLC and SLN were observed on HS68 fibroblast cells after exposure to UV.

 

# Line 322 says that using IDE did not increase the hydration ability of either SNL or NLC. Is this not contradictory to what is envisioned?

Answer

It is unclear why this statement is contradictory, as we did not claim that an increase of skin hydration was expected due to topical treatment with gels containing IDE loaded SLN and NLC. IDE is a well-known antioxidant agent but its hydrating ability has not been clearly demonstrated. We investigated the hydrating effect of gels containing unloaded and IDE loaded SLN and NLC and the results of our study revealed that, under the experimental conditions used in this work, IDE did not show any hydrating effect. 

 

# Authors are providing too many references. Only the most relevant ones need to be included. Also, instead of frequently referring to what has been done by the authors in the past studies, it is more important to precisely focus on the current study.

Answer

All references we cited had been included to provide the reader with references that report useful information and further details about the background, protocol, experimental procedures and techniques we used in the present manuscript. In addition, the references we have added about our previous works point out that we have an expertise in the topic of the present manuscript and that we have already published in renowned journals the protocol, the experimental procedures and the techniques we used in the present manuscript. Therefore, the cited references cannot be regarded as irrelevant.

 

Author Response File: Author Response.pdf

Reviewer 2 Report

The work is interesting and shows some translational potential, however the manuscript needs to be improved in terms of organization, data presentation and discussion. Some parts are unclear and some experimental errors impact manuscript quality and therefore it cannot be accepted in the present form but a major revision is advised before being reconsidered for publication.

Major:

1) Some important characterization is missing. First, the actual IDE and Vit E contents should be determined. The AA have reported only the theoretical loading and not the actual one. Second, stability has been measured only looking at size and surface charge, no potential drug losses have been evaluated. Lipid nanoparticles are well known for their tendency to provoke drug expulsion over time due to polymorphism. Therefore, in order to state that such formulations are stable, drug content should be measured upon storage.

2) The in vivo clinical study is a little puzzling and superficially conducted. First, the AA stated that no approval was necessary for their protocol. However, this cannot be correct since the protocol is still a clinical study and as a general rule an evaluation and approval from the ethical committee is required anyway. By law, a ministry evaluation may also be needed in case of safety concerns. I suggest to rephrase their statement. 

Moreover, what kind of protocol is this one? Safety? Compliance? Efficacy? It should be clarified. The AA have evaluated the effect on pigmentation as a surrogate for the protective efficacy of the formulations. However, in order to normalize such an effect, they should have considered exposure levels of volunteers to light during the testing period. Why has it not been considered? Perhaps instructing the volunteers in following specific behaviors and habits during the study? In addition, how individual differences such as age or skin sensitivity have been normalized? How has the recruitment been conducted? Any exclusion criteria?

3) Regardless IDE or Vit E solubility, gels with free IDE and IDE + Vit E should be tested as controls in the in vivo study, otherwise the protocol is not complete. Suspensions rather than solutions are conceivable.

4) Figure 5 is unclear. The way it is connected to the experimental section 2.10 is not understood. In that section, the AA state that all formulations were tested at the same concentration of 25 micro/mL, but in fig. 5 there are four increasing concentrations for each formulation and controls. Explain and in case correct the experimental description. 

5) Table 2 should be better discussed. There seem to be a certain correlation between PIT and size of SLN, how can it be explained? The addition of Vit E seems to break up such a correlation and PIT decreases with IDE loading. Explain.

Minor:

1) TEM images should be better resolved and at higher magnification. How the AA have avoided artifacts having not used cryogenic techniques?

2) In figure 2 thermograms of the formulations at 1.5% IDE loading should be reported as well.

3) What about IDE solubility in Vit E? have the AA evaluated it? it could be important to characterize SLN vs NLC behavior.

4) In section 2.10 title the AA declare to evaluate the antioxidant activity. It is not correct, as what investigated in section 2.10 is only the protective effect of the formulations upon irradiation of cell cultures. No ROS expression or other parameters have been measured.  Correct it.

Author Response

Reviewer 2

The work is interesting and shows some translational potential, however the manuscript needs to be improved in terms of organization, data presentation and discussion. Some parts are unclear and some experimental errors impact manuscript quality and therefore it cannot be accepted in the present form but a major revision is advised before being reconsidered for publication.

 

Major:

 

1) Some important characterization is missing. First, the actual IDE and Vit E contents should be determined. The AA have reported only the theoretical loading and not the actual one. Second, stability has been measured only looking at size and surface charge, no potential drug losses have been evaluated. Lipid nanoparticles are well known for their tendency to provoke drug expulsion over time due to polymorphism. Therefore, in order to state that such formulations are stable, drug content should be measured upon storage.

Answer

As reported in literature (Jenning V, Schäfer-Korting M, Gohla S. Vitamin A-loaded solid lipid nanoparticles for topical use: drug release properties. J Control Release. 2000, 15, 66, 115-26), when water insoluble compounds are incorporated into lipid nanoparticles, if the resulting colloidal dispersion is clear, all drug must be in the lipid phase of the SLN dispersion. Therefore, in clear colloidal dispersion, “the incorporation rate is approximately 100%”. In a previous work (see reference 21), we determined that IDE water solubility was 5 µg/ml. Therefore, when we obtained clear lipid nanoparticles dispersion the maximum amount of IDE that could be found in the water phase was 5 µg/ml. We loaded at least 1% w/w IDE in these lipid nanoparticles. Therefore, IDE concentration in the lipid nanoparticle colloidal dispersion, expressed as µg/ml, was 10000 µg/ml. As we obtained clear colloidal dispersion at this IDE concentration, the maximum percentage of IDE in the water phase could be 0.05 % of total amount used to obtain the colloidal dispersion. To the best of our knowledge, the accuracy of a common analytical method such as HPLC is about 0.1%. Therefore, the amount of IDE in the water phase of our clear colloidal dispersions was lower than the standard error of an HPLC method. Accordingly, it is pointless to determine by an analytical method the amount of drug loaded into lipid nanoparticles when a great amount of water insoluble drugs is loaded in lipid nanoparticles that produce clear colloidal dispersion as drug loading could be considered approximately 100%.

We agree with the reviewer that one of the major drawbacks of lipid nanoparticles is drug leakage during storage. However, during storage of our lipid nanoparticle samples we did not notice any sign of precipitation and our samples remained clear after the storage period. Therefore, no drug leakage occurred during storage. Due to the reviewer’s comment, we realized that we omitted to specify that we did not observe any sign of drug precipitation during storage. Therefore, we added in the text the following sentence (line 273 revised version): In addition, we did not observe any sign of drug precipitation during storage.

 

2) The in vivo clinical study is a little puzzling and superficially conducted. First, the AA stated that no approval was necessary for their protocol. However, this cannot be correct since the protocol is still a clinical study and as a general rule an evaluation and approval from the ethical committee is required anyway. By law, a ministry evaluation may also be needed in case of safety concerns. I suggest to rephrase their statement.

Answer

We did not perform a clinical study and we have never claimed in the manuscript that the in vivo protocol we used was a clinical study. The ethical committee declared that no approval was required to perform this in vivo study because this type of protocol: a) does not require to be supervised by a physician; b) does not involve invasive procedures; c) use topical products whose safety has been clearly demonstrated; d) can be performed in a beauty center as it is an “in use” test of products that can be used for cosmetic purposes.

In addition, we have already published previous works (see references 11, 12, 28) describing the same protocol and reporting that the ethical committee declared that no approval was required to perform this type of in vivo study.

 

 

Moreover, what kind of protocol is this one? Safety? Compliance? Efficacy? It should be clarified. The AA have evaluated the effect on pigmentation as a surrogate for the protective efficacy of the formulations. However, in order to normalize such an effect, they should have considered exposure levels of volunteers to light during the testing period. Why has it not been considered? Perhaps instructing the volunteers in following specific behaviors and habits during the study? In addition, how individual differences such as age or skin sensitivity have been normalized? How has the recruitment been conducted? Any exclusion criteria?

Answer

As clearly highlighted in the title of the manuscript, we studied the in vivo topical efficacy of lipid nanoparticles co-loading idebenone and tocopheryl acetate. Therefore, the in vivo protocol was used to evaluate the efficacy of the formulations under investigation. However, to better point out that we assessed the efficacy of these formulations, in the materials and methods section, we modified the text as follows (line 237 revised version): In vivo topical efficacy under in use conditions of unloaded and IDE loaded SLN and NLC was assessed according to a protocol previously described.

The protocol we used is an in vivo study under in use conditions. Therefore, the volunteers were not asked to refrain from their normal habits. As reported in the experimental section, baseline values were determined for each subject prior to the application of the formulations under investigation and results were expressed as difference between the initial and final value. We did not observe any influence of the subject age on the results we obtained. Subjects with skin sensitivity problems were not admitted in the study. As reported in the manuscript, only healthy volunteers were recruited.  We selected a panel of healthy volunteers with normal/moderate dry skin. Young healthy subjects (mean age 30-40 years) were not admitted in the study because they showed very high hydration baseline values (indicating fully hydrated skin) that made them unsuitable to evaluate the effects of skin hydrating treatments.

 

3) Regardless IDE or Vit E solubility, gels with free IDE and IDE + Vit E should be tested as controls in the in vivo study, otherwise the protocol is not complete. Suspensions rather than solutions are conceivable.

Answer

Drug percutaneous absorption is a passive process that depends on the drug concentration gradient between the surface and the inner layers of the skin. Therefore, drug concentration in the vehicle plays an important role in determining the amount of drug that penetrated into the skin. Saturated vehicles have the maximum thermodynamic activity and provide the maximum driving force for the percutaneous absorption process for a specific vehicle. As IDE is poorly water soluble, its incorporation in a hydrophilic gel at the same percentage loaded into lipid nanocarriers (1 or 1.5% w/w) would result in a suspension. In this formulation, IDE would be at the maximum thermodynamic activity while its thermodynamic activity could be lower in gel containing SLN or NLC, being IDE incorporated in the lipid core. Therefore, the use of such formulation as control could underrate (or overrate) the effects of our samples.

Vitamin E acetate is a poorly water-soluble compound, as well. Vitamin E acetate incorporation in a hydrophilic gel would result in a phase separation, making this vehicle unsuitable for any practical application.  

Therefore, we did not use gels containing free IDE and free IDE and vitamin E acetate as control.

In addition, the aim of our study was to compare the topical effects of lipid nanoparticles loaded only with IDE or co-loading IDE and vitamin E acetate. The comparison between these lipid nanocarriers and conventional vehicles containing free IDE and/or vitamin E acetate was out of the scope of our work.

 

4) Figure 5 is unclear. The way it is connected to the experimental section 2.10 is not understood. In that section, the AA state that all formulations were tested at the same concentration of 25 micro/mL, but in fig. 5 there are four increasing concentrations for each formulation and controls. Explain and in case correct the experimental description.

Answer

We thank the reviewer for the comment. We amended the text (line 207 revised version) as follows to better detail the experimental protocol we used: “Different concentrations of IDE (10-100 µg/ml dissolved in 0.1% ethanol) were added to previously seeded cells on a 96 well plate and left to incubate for 24 hours and afterward the viability of cells was assessed. Subsequently, cells were treated for 24 hours with an amount of IDE loaded SLN and NLC corresponding to the highest IDE concentration that did not lead to cytotoxic effects (25µg/ml).” Accordingly, we modified Fig. 5 and inserted a new Fig. 6. Therefore, in the revised version:

Fig. 5 shows the      effect of increased doses of idebenone (IDE) loaded solid lipid      nanoparticles (SLN) and nanostructured lipid carriers (NLC) on the      percentage of vitality of HS68 fibroblast cells, determined by MTT

Fig. 6      illustrates the effect of IDE loaded SLN and NLC (25 µg/ml) on the      vitality percentage of HS68 fibroblast cells exposed to UV radiation

The results depicted in Fig. 6 are reported at line 471 in the revised version.

 

5) Table 2 should be better discussed. There seem to be a certain correlation between PIT and size of SLN, how can it be explained? The addition of Vit E seems to break up such a correlation and PIT decreases with IDE loading. Explain.

Answer

As the reviewer believed that “there seem to be a certain correlation between PIT and size of SLN”, we plotted SLN size vs PIT values to verify the existence of a relationship between these two parameters.

We obtained the following plot:

Looking at the plot reported above, it is evident that no correlation exists between SLN size and PIT values.

In addition, it has no statistical meaning to perform a linear regression analysis having only three data points.

 

Minor:

1) TEM images should be better resolved and at higher magnification. How the AA have avoided artifacts having not used cryogenic techniques?

Answer

In a previous work, we submitted TEM images that having higher magnification showed few nanoparticles. We were asked to report TEM images where at least ten/fifteen nanoparticles were shown to account for particle size distribution. Therefore, due to our previous experience, in this manuscript we reported TEM images with lower magnification but showing at least ten nanoparticles to highlight particle size distribution. TEM images originate from the equipment used and they have the best resolution we could obtain translating them into the manuscript template.  We have already used the same TEM technique described in this manuscript to obtain morphological information about lipid nanocarriers (see references 11, 12, 17, 20, 21, 23) and our images are similar to those reported in literature by other authors who used the same TEM technique; therefore, no artifact could be supposed to occur.

 

2) In figure 2 thermograms of the formulations at 1.5% IDE loading should be reported as well.

Answer

To comply with the reviewer’s request, we inserted the thermograms of lipid nanocarriers loaded with 1.5% w/w IDE. However, to avoid thermogram overlapping, we splitted Fig. 2 into two panels. Panel a) shows the thermograms of unloaded and IDE loaded SLN; panel b) shows the thermograms of unloaded and IDE loaded NLC.

 

3) What about IDE solubility in Vit E? have the AA evaluated it? it could be important to characterize SLN vs NLC behavior.

Answer

We evaluated IDE solubility in VitE. IDE solubility in VitE was greater than 100 mg/g. Therefore, according to the U.S. Pharmacopoeia, IDE is freely soluble in VitE. As reported in the manuscript (line 320 revised version), the presence of the oil component (VitE) could favor IDE solubilization in the lipid matrix of the nanoparticles, thus increasing IDE loading in comparison to SLN.

 

4) In section 2.10 title the AA declare to evaluate the antioxidant activity. It is not correct, as what investigated in section 2.10 is only the protective effect of the formulations upon irradiation of cell cultures. No ROS expression or other parameters have been measured.  Correct it.

Answer

We thank the reviewer for the comment. We amended the text as follows:

2.10. Evaluation of cytotoxicity and photo-protective effect in fibroblast cell line HS-68

3.4. Evaluation of cytotoxicity and photo-protective effect in fibroblast cell line HS-68

 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Thank you for addressing the concerns in full details. The manuscript has attained much better clarity with the changes you have included. I would recommend it for publication as it is. 

Author Response

We thank the reviewer for reviewing our manuscript.

Reviewer 2 Report

The answers given to the concerns arose from the first reviewing of the manuscript are not adequate and absolutely not scientifically acceptable as formulation properties cannot be inferred based on other publications this is an absolute non sense.

In addition I believe that novelty of the work is a concern, unless justified by the authors. See the work of Li et al. Nanostructured lipid carriers improve skin permeation and chemical stability of idebenone AAPS PharmSciTech 2012, 13,1. This work shows better how such formulations should be characterized measuring content and content stability of idebenone, steps that have not been done in this manuscript. This work was not cited in the manuscript but is an important precedent and the authors need to highlight the differences in order to demonstrate the actual novelty of their work beyond just the use of different excipients.

Moreover, the other concern regards the in vivo efficacy study that the authors claim not being a clinical study. I recommend a careful check on this point as albeit guidelines are not completely clear and exhaustive on the use this kind of topical formulations, there are plenty of papers in which similar formulations have been clinically tested (even idebenone creams, see the works of McDaniel and many others, see even some skin care products). The authors' justification based on their previous published works is not convincing as, even though there are several marketed topical products of idebenone, they are proposing an efficacy study not on a marketed product, but on a supposedly new nanoformulation that is likely to require clinical settings and, contrarily to their claim, such studies should not be allowed in a beauty center. In this regard, toxicity is also a concern as even when using GRAS excipients the safety profile of the formulation should be determined especially considering some evidences of idebenone-induced dermatitis. 

in light of all such issues, I do not find the work of a sufficient scientific level to justify publication, unless the authors are willing to perform the required studies and clear the concerns regarding novelty and present the required documentation demonstrating the non-clinical status of their study.

Author Response

Reviewer 2

The answers given to the concerns arose from the first reviewing of the manuscript are not adequate and absolutely not scientifically acceptable as formulation properties cannot be inferred based on other publications this is an absolute non sense.

Answer

In this work, we did not infer the properties of our lipid nanoparticles based on other publications. We experimentally determined size, polydispersity index and zeta potential. In addition, we performed DSC analyses and we evaluated the morphology of these nanoparticles by TEM.  We based the determination of the amount of drug loaded in these nanoparticles on the application of the physico-chemical laws governing drug solubility. If we have a drug whose water solubility (at room temperature) is 5 µg/ml and we put 10 mg of this drug in 1 ml of water, 5 µg of the drug will be in solution while the remaining 9995 µg will precipitate. Therefore, we do not need to determine the content of drug in solution or in the precipitate because our knowledge of the laws governing drug solubility provide us this information. We applied this physico-chemical law to determine the amount of drug contained in our lipid nanoparticles. We used 10 mg/ml or 15 mg/ml of idebenone (IDE, water solubility 5 µg/ml) to prepare our lipid nanoparticles and we obtained clear vehicles. Therefore, the physico-chemical laws governing drug solubility tell us that maximum 5 µg of IDE were in solution while the remaining 9995 µg (when we used 10 mg/ml) or 14995 (when we used 15 mg/ml) must be inside the nanoparticles.  Therefore, if loading a great amount of water insoluble drugs into lipid nanoparticles produces a clear colloidal dispersion, drug loading could be considered approximately 100%. Other authors have applied the same concept to determine the amount of water insoluble drugs into lipid nanocarriers (see Jenning V, Schäfer-Korting M, Gohla S. Vitamin A-loaded solid lipid nanoparticles for topical use: drug release properties. J Control Release. 2000, 15, 66, 115-26). We applied the same concept in previous papers (see references 17, 21, 23, 28, 33) studying IDE loaded solid lipid nanoparticles (SLN) and we have never been asked to perform experimentally this determination. If we determined the amount of drug in solution and/or inside the nanoparticles, it would mean that we do not know or we do not understand the physico-chemical laws governing drug solubility. However, before publishing our first work on IDE loaded SLN (Montenegro et al., In vitro evaluation of idebenone-loaded solid lipid nanoparticles for drug delivery to the brain. Drug Dev. Ind. Pharm. 2011, 37, 737–746 DOI: 10.3109/03639045.2010.539231) we experimentally determined the amount of IDE loaded into SLN to check if this concept was applicable to our nanoparticles. We found that the amount of IDE loaded into SLN was 99.8 ± 0.9 %. Therefore, we decided to avoid reporting the experimental determination and we described the application of the drug solubility concept that makes pointless the experimental determination of such parameter in the conditions of our study.

If this reviewer believes that applying our knowledge of the physico-chemical laws is scientifically incorrect, we should conclude that many works should not have been published because the authors did not experimentally determine and measure all data they reported in the work. 

The reviewer cited as example of IDE loading experimental determination the work published by Li and Ge in 2012. About one year ago, we published a review on the strategies to improve IDE bioavailability (see reference 16) where we cited the work published by Li and Ge. Therefore, we read this paper and we knew the data reported in this manuscript. As the reviewer could notice reading the work published by Li and Ge, the amount of IDE loaded into nanostructured lipid carriers (NLC) was 99.4±0.8. Therefore, these authors found that the amount of IDE entrapped into NLC was approximately 100%. However, the goal of the manuscript published by Li and Ge was “to develop a carrier system that is able to improve the chemical stability of IDB (idebenone) and enhance the skin delivery of IDB”. In particular, the authors compared the stability of three different formulations, i.e., IDE loaded NLC, IDE loaded nanoemulsion, and IDE dissolved oil solution. As the aim of the work by Li and Ge was to evaluate the stability of these vehicles and IDE contained in these vehicles under different storage conditions ( 25°C in the dark, 25°C in the light, 40°C 75% relative humidity), they necessarily had to determine the amount of IDE in their carriers experimentally.

In the first work we published on IDE loaded SLN (Montenegro et al., In vitro evaluation of idebenone-loaded solid lipid nanoparticles for drug delivery to the brain. Drug Dev. Ind. Pharm. 2011, 37, 737–746 DOI: 10.3109/03639045.2010.539231), we determined the stability of our IDE nanoparticles under different storage conditions (21°C in the dark and 37°C in the dark). As reported in that manuscript, we did not detect any significant change in SLN properties (size, polydispersity index and zeta potential) when they were stored at 21°C. On the contrary, they were unstable when stored at 37°C.  At the end of the storage period at 21°C, we experimentally determined the amount of IDE entrapped in the nanoparticles. As expected (SLN properties such as size, polydispersity index and zeta potential were unchanged and the samples remained clear with not sign of precipitation), we did not find any significant change in comparison with freshly prepared samples. Therefore, as it was obvious that no change of IDE entrapment could be occurred because the properties of the nanoparticles were unchanged, we did not report the experimental determination of IDE entrapment at the end of the storage period. Obviously, in the following works, we did not repeat stability studies at 37°C but we checked only the stability at 21°C to confirm the nanocarrier stability. Therefore, in the present work, we assessed the stability of our lipid nanoparticles only at 21°C in the dark. We did not find any significant change in lipid nanoparticle properties and visual aspect during storage and we reported in the text “they proved stable after storage for two months at room temperature as no change of particle size and PDI values were observed (data not shown). In addition, we did not observe any sign of drug precipitation during storage”.

 

In addition I believe that novelty of the work is a concern, unless justified by the authors. See the work of Li et al. Nanostructured lipid carriers improve skin permeation and chemical stability of idebenone AAPS PharmSciTech 2012, 13,1. This work shows better how such formulations should be characterized measuring content and content stability of idebenone, steps that have not been done in this manuscript. This work was not cited in the manuscript but is an important precedent and the authors need to highlight the differences in order to demonstrate the actual novelty of their work beyond just the use of different excipients.

Answer

As mentioned above, the goal of the manuscript published by Li and Ge was “to develop a carrier system that is able to improve the chemical stability of IDB (idebenone) and enhance the skin delivery of IDB”. These authors used a synthetic oil with no intrinsic pharmacological activity (medium chain fatty acid triglycerides) to prepare NLC and nanoemulsions and they determined in vitro IDE permeation from these nanocarriers through guinea pig skin. Our work investigated the effect of co-loading two antioxidants into lipid nanoparticles to evaluate if they had a synergic effect. We co-loaded Idebenone and vitamin E acetate. As vitamin E acetate is an oil, the resulting nanocarriers where NLC while the nanocarriers loading only IDE were SLN. Therefore, as reported in the text of the manuscript, we used an oil with intrinsic pharmacological activity. To the best of our knowledge, to date no other work has investigated the effect of co-lading IDE with another antioxidant into lipid nanoparticles to improve the antioxidant effect. We compared the technological properties, in vitro antioxidant and photo-protective effect, in vivo topical effects on human volunteers of IDE loaded SLN and IDE loaded NLC. Therefore, the goal and the protocol of our work were very different from the goal and the protocol of the paper published by Li and Ge. If the reviewer believes that we merely changed excipients then we should infer that, most works in the field of pharmaceutical technology lack of novelty because the authors investigate drug bioavailability from carriers and vehicles that differ from previously published ones only because of the excipients used in their preparation or because of a different technique of preparation. In addition, vitamin E acetate is an active ingredient and cannot be regarded as an excipient.

The reviewer believes that we should compare our works with that published by Li and Ge. As mentioned above, Li and Ge compared IDE loaded NLC with IDE loaded nanoemulsion. The composition of IDE loaded NLC prepared by Li and Ge is completely different from that we used in our work. In addition, Li and Ge performed in vitro skin permeation experiments on guinea pigs while we carried out in vivo studies to evaluate the topical effects of IDE loaded SLN and NLC on human volunteers. Therefore, it is unclear which kind of comparison we were supposed to make between our work and the work by Li and Ge.

In addition, Li and Ge reported that they studied the chemical stability of IDE after inclusion in NLC and nanoemulsion. The authors observed a significant decrease of IDE content in nanoemulsion that they attributed to IDE decomposition without supporting this hypothesis with any experimental evidence. Moreover, the authors found that the initial IDE content in nanoemulsion (96.2±1.1) fell rapidly and it had already declined to 67.2% (25°C, dark) and 52.4% (25°C, daylight) at the end of 180-day storage, respectively. As IDE is water insoluble, if it leaked out of the nanocarrier in such high percentage it would precipitate but the authors did not report that they observed any precipitate. Several dark sides make the scientific soundness of this work questionable. Therefore, we decided not to cite this work in our manuscript.  

 

Moreover, the other concern regards the in vivo efficacy study that the authors claim not being a clinical study. I recommend a careful check on this point as albeit guidelines are not completely clear and exhaustive on the use this kind of topical formulations, there are plenty of papers in which similar formulations have been clinically tested (even idebenone creams, see the works of McDaniel and many others, see even some skin care products). The authors' justification based on their previous published works is not convincing as, even though there are several marketed topical products of idebenone, they are proposing an efficacy study not on a marketed product, but on a supposedly new nanoformulation that is likely to require clinical settings and, contrarily to their claim, such studies should not be allowed in a beauty center. In this regard, toxicity is also a concern as even when using GRAS excipients the safety profile of the formulation should be determined especially considering some evidences of idebenone-induced dermatitis.

Answer

As the reviewer pointed out, there are no specific guidelines for testing the safety and efficacy of cosmetic products. According to the European legislation, clinical tests on cosmetic products are not mandatory. Therefore, cosmetic products can be marketed without performing clinical tests but their safety and efficacy can be demonstrated using a suitable protocol, depending on the type of product under evaluation.

Clinical tests must be performed under the supervision of a physician. The reviewer cited the work performed by McDaniels DH, Neudecker BA, DiNardo JC, Lewis II JA and Maibach HI, Journal of Cosmetic Dermatology, 2005, 4, 167–173.  The author McDaniels is a physician and Howard I Maibach is one of the most famous dermatologist all over the world. Being physicians, they can perform clinical studies. However, reading the manuscript, it is evident that this study was not authorized or approved by any ethical committee although invasive procedures (skin biopsies) were involved. 

None of the authors of the manuscript under review is a physician. Therefore, we could not perform a clinical trial without asking a physician to supervise the study. In the text of our manuscript, we did not report that the in vivo study was supervised by a physician. Therefore, our study cannot be regarded as a clinical study. As we reported in our previous answer, the ethical committee declared that no approval was required to perform this in vivo study because of the type of protocol we used. The decision of the ethical committee was based on papers already published in literature reporting similar protocols (such as the work by McDaniels et al.) where no approval by an ethical committee was indicated.

At present, as the reviewer noticed, some topical products containing idebenone are commercialized as cosmetic products. According to the European legislation, cosmetic products must be safe for the consumer. Cosmetic products cannot be launched on the market without being tested for their safety. Therefore, the reviewer’s concern about idebenone toxicity is not justified.

in light of all such issues, I do not find the work of a sufficient scientific level to justify publication, unless the authors are willing to perform the required studies and clear the concerns regarding novelty and present the required documentation demonstrating the non-clinical status of their study.

Answer

We welcome every constructive criticism that could allow us improving the quality of our work. However, we would like to point out that, in the first round of the reviewing process, this reviewer raised several baseless criticisms. For example, the reviewer stated “There seem to be a certain correlation between PIT and size of SLN, how can it be explained? The addition of Vit E seems to break up such a correlation and PIT decreases with IDE loading. Explain.” The reviewer found a relationship that did not exist and asked us to explain this non-existing relationship. The reviewer stated, “Regardless IDE or Vit E solubility, gels with free IDE and IDE + Vit E should be tested as controls in the in vivo study, otherwise the protocol is not complete”. We were asked to use as controls formulations in which a phase separation would occur. No one would use such formulations because the non-homogenous dispersion of the active ingredients would make these vehicles unsuitable for any practical application.

Author Response File: Author Response.pdf