Review Reports - Dependence of Viscosity and Diffusion on <i>β</i>-Cyclodextrin and Chloroquine Diphosphate Interactions

Round 1

Reviewer 1 Report

The authors of the manuscript report on the study of the diffusion of the mixture of small and large molecules. The reported experiments seem well-conducted, but the data analysis is the major concern for this reviewer. It would be nice to see the Taylorgram and the fit. Equation 2 should be explained since the reference [2] is in a hard-to-find journal. The legend of equation 2 should include “r” (tube radius?). Equations 5-14 should be explained, or referenced better since the current references are not helpful. What is the sense of negative diffusion coefficients? The sense of the equations 19-22 is not clear and should be explained. Due to such significant questions to the data analysis, the reviewer suggests a major revision of the paper.

Author Response

Reply to Reviewers’ comments

Manuscript ID: 1309861

Title: Effect of the carrier β-cyclodextrin on the transport properties of
chloroquine diphosphate in aqueous solutions

Authors: Lenka Musilová, Aleš Mráček, Ana C.F. Ribeiro, Eduarda F. G. Azevedo, M. Melia Rodrigo, Artur J.M. Valente, Miguel A. Esteso

Reviewer #1

We are grateful for these comments. Concerning the data analysis, the referee is right and, consequently, we have inserted the Taylorgram and the text.

Equation 2 should be explained since the reference [2] is in a hard-to-find journal.

The referee is right and, consequently, we have inserted the text accordingly.

The legend of equation 2 should include “r” (tube radius?).

We really sorry for that. We have inserted the text accordingly. That is, r represents the inner radius of the dispersion tube.

Equations 5-14 should be explained, or referenced better since the current references are not helpful.

The referee is right and, consequently, we have inserted the text accordingly.

What is the sense of negative diffusion coefficients?

We are grateful in advance for the question raised by the referee. In fact, the carrier-mediated transport phenomena in multicomponent systems it is not trivial one of the physical-chemistry. Despite the answer of this question is done on page 7, line 218 (Nevertheless, because the D₂₁ values are almost zero, the gradient in the concentration of CDP produces weak counter-current coupled flows of β-CD), we recognize that given the complexity of this area, we must provide a more detailed explanation, having in mind its clarification.

Having in mind the equations

J₁ = -D₁₁ÑC₁- D₁₂ÑC₂

J₂ = -D₂₁ÑC₁- D₂₂ÑC₂

non-zero values of cross diffusion terms D_ik indicate interactions between the fluxes of solutes 1 and 2. A negative cross coefficient diffusion (D_ik) indicates counter-current coupled transport of solute i from regions of lower to higher concentration of solute k. In our particular case ({CDP(C₁) + b-CD(C₂)}), it was observed that for some concentrations of CDP, D₂₁ <0. These negative D₂₁ values indicate that CDP gradients can drive counter current coupled flows of b-CD towards regions of higher CDP.

The sense of the equations 19-22 is not clear and should be explained.

The referee is right and, consequently, we have modified some parts and, in addition, we have inserted the text accordingly. Once this model is well described in references [32-35], and also to avoid repetitions and also some plagiarism, only some parts are indicated in the text.

Author Response File: Author Response.pdf

Reviewer 2 Report

In this work, Musilova et al. described the influence of viscosity and binary and ternary mutual diffusion coefficient value of different systems with and without cyclodextrin. Moreover, the authors carefully presented the changes in binary and ternary mutual diffusion coefficients. However, to improve the scientific quality of the manuscript, the authors should perform some new experiments or add some new phrases:

The author should explain why they used the hydrophilic drug for the formation of the inclusion complex in more detail. It is known that only hydrophobic drugs can be entrapped into the hydrophobic cyclodextrin cavity. Therefore, the authors should confirm the formation of the inclusion complex between βCD and CDP by using additional methods for example described in Materials Today Communications 25 (2020) 101605.
The title is not adequate to the content contained in the manuscript. The title should be changed.
In the introduction, the authors mentioned only the CDP and its solubility. In this section, the authors should discuss the influence of assigned values on drug transport in more detail.
The structure of CD and CDP should be added to the manuscript.
The authors should remove the parentheses, for example, line 80 page 2. It should be 298,15+0,01 without parentheses. It should be corrected in the whole manuscript.
The dots between mol and dm³ or mPa and s should be removed (Table 2).
The authors should evaluate the mutual diffusion coefficient value in lower and higher temperatures. The low temperature can facilitate the formation of the inclusion complex. Is it possible to remove the salt from CDP and perform the measurement for free Chloroquine? This process should result in hydrophilization of CDP and better encapsulation inside the βCD cavity.
In conclusion, the authors should explain the novelty and significant influence on drug delivery development.

Comments for author File: Comments.pdf

Author Response

Reviewer#2

The author should explain why they used the hydrophilic drug for the formation of the inclusion complex in more detail. It is known that only hydrophobic drugs can be entrapped into the hydrophobic cyclodextrin cavity. Therefore, the authors should confirm the formation of the inclusion complex between βCD and CDP by using additional methods for example described in Materials Today Communications 25 (2020) 101605.

We thank the reviewer for this suggestion; in fact, 1H and 2D NMR or and/ molecular dynamics are useful techniques to validate the occurrence of host-guest interactions; in fact, we have done that in some of our previous papers. However, this is not the case hence the formation of inclusion compounds has already been proved and reported by other authors. In order to clarify this point the following phrase has been added. “Recently, it has been demonstrated by NMR and computational studies that chloroquine is able to protrude both α- and β–cyclodextrins, being that the stronger interaction occurs with the β-cyclodextrin [14].”

A new reference has been added (14. https://doi.org/10.1016/j.molstruc.2020.129696), and the references have been renumbered throughout the ms.

The title is not adequate to the content contained in the manuscript. The title should be changed.

The title has been modified to “Dependence of viscosity and diffusion on β-cyclodextrin and chloroquine diphosphate interactions”

In the introduction, the authors mentioned only the CDP and its solubility. In this section, the authors should discuss the influence of assigned values on drug transport in more detail.

The 2^nd paragraph of the Introduction has been changed and now reads:

CDP is soluble in water and has a high bioavailability when administrated orally [4]. However, this drug also shows some significant side effects [7]. These can be reduced by forming host-guest supramolecular compounds with cyclodextrins [2]. It is known that due to the amphiphilic behavior of cyclodextrins, host-guest interactions mainly occur in-side the hydrophobic CD’s cavity with hydrophobic guests [8,9]. However, interactions between guest molecules and cyclodextrins can also occur via, for example, H-bonding involving hydroxyl groups located outside the CD’s cavity [10,11]. Despite its solubility …”

More four references (8,9, 10 and 11) have been added

The structure of CD and CDP should be added to the manuscript.

A Scheme with the structures of both compounds was added

The authors should remove the parentheses, for example, line 80 page 2. It should be 298,15+0,01 without parentheses. It should be corrected in the whole manuscript.

We have followed the reviewer’s suggestion.

The dots between mol and dm³ or mPa and s should be removed (Table 2).

Dots have been removed as suggested.

The authors should evaluate the mutual diffusion coefficient value in lower and higher temperatures. The low temperature can facilitate the formation of the inclusion complex. Is it possible to remove the salt from CDP and perform the measurement for free Chloroquine? This process should result in hydrophilization of CDP and better encapsulation inside the βCD cavity.

The reviewer is absolutely right on the effect of temperature on the host-guest equilibrium; however, temperature effects, such as activation energies, are not part of the study.

In conclusion, the authors should explain the novelty and significant influence on drug delivery development.

This is a good point; interdiffusion coefficients reported here correspond to the maximum value of diffusion (or rate constant, if we assume a Fickian drug delivery process –see, for example, J. Crank. The Mathematics of Diffusion) in a drug delivery process. Consequently, it can (or it must) be seen as a reference value. Unfortunately, it is not common to obtain this value from the literature due to the very strict experimental conditions required to measure this physical property. On the other hand, viscosity (in this case Newtonian viscosity) is quite relevant for different kind of formulations and also to understand if changes in diffusion coefficients results from alteration in the viscosity of solution or due to modification of the structure of the solution. At the end of the section 1, the following statement has been added: “Additionally, interdiffusion coefficients correspond to the maximum limit value for the release kinetics of drugs (or complexes), in drug delivery systems.”

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

This reviewer is partially satisfied with the authors’ responses. Regarding equations 22-25, authors should put mentioned mathematical rearrangements in the supplemental material, so any reader can follow. Figure 1 needs to be better prepared and explained. The fitting line is not visible. What is v in the V(t)/v? It seems that equation 2 does not describe differential detection, please explain how experimental data were fitted. There are few typos in the revised text, for example, line 124 “a(t)composition”, and line 275 “the is the sum”.

Author Response

Reply to Reviewers’ comments

Manuscript ID: 1309861

Title: Effect of the carrier β-cyclodextrin on the transport properties of
chloroquine diphosphate in aqueous solutions

Authors: Lenka Musilová, Aleš Mráček, Ana C.F. Ribeiro, Eduarda F. G. Azevedo, M. Melia Rodrigo, Artur J.M. Valente, Miguel A. Esteso

Manuscript ID: 1309861

Title: Effect of the carrier β-cyclodextrin on the transport properties of
chloroquine diphosphate in aqueous solutions

Authors: Lenka Musilová, Aleš Mráček, Ana C.F. Ribeiro, Eduarda F. G. Azevedo, M. Melia Rodrigo, Artur J.M. Valente, Miguel A. Esteso

Reviewer #1

We are grateful for these comments.

Concerning the equations 22-25, the referee is right and, consequently, we have inserted additional information in the supplementary material.

Figure 1 needs to be better prepared and explained. The fitting line is not visible. What is v in the V(t)/v? It seems that equation 2 does not describe differential detection, please explain how experimental data were fitted. There are few typos in the revised text, for example, line 124 “a(t)composition”, and line 275 “the is the sum”.

The referee is right and, consequently, we have improved the figure 1 and the respective caption. Concerning the meaning of V, it represents the unit of the refractometer voltage in volt (V). The figure indicates the ternary dispersion profile (the refractometer voltage in volt (V) against time) generated by two injections. That is,

a) a 0.063 cm³ sample of chloroquine diphosphate 005 mol dm⁻³into 0.007 mol dm⁻³ β-cyclodextrin (Profile ∆C₁ = 0.005 mol dm⁻³, ∆C₂ = 0);
b) a 0.063 cm³ sample of 002 mol dm⁻³β-cyclodextrininto 0.007 mol dm⁻³ β-cyclodextrin

(Profile ∆C₁ = 0 mol dm⁻³, ∆C₂ =0.002 mol dm⁻³). Measured (o) and fitted (-, eq. 5)

Author Response File: Author Response.pdf

Reviewer 2 Report

The authors corrected the manuscript according to the reviewer’s suggestion, and now, the quality and presentation of the results are improved. The authors added the new references, however, these references do not support the reviewer’s previous comments. I recommend this article for publication in Processes after additionally comments:

The authors added the new sentence: “Recently, it has been demonstrated by NMR and computational studies that chloroquine is able to protrude both α- and β–cyclodextrins, being that the stronger interaction occurs with the β-cyclodextrin [14].” the NMR data, only the computational studies. I had to find the extra references in the cited article that contained the information about NMR data. However, Roy et al. performed only the 1H NMR spectrum. They claimed that the shifting of ring protons is enough to prove the formation of the inclusion complex. The 1H NMR spectrum can only provide information about the creation of some interactions. The 1H NMR spectrum does not provide information about the types of interaction. I am not convinced that the highly hydrophobic chloroquine diphosphate possesses the ability to the formation of inclusion complex with a hydrophobic cyclodextrin cavity. Additionally, authors claimed that “For more concentrated solutions, for 314 which it is obtained that D12>0, a co-flow of CDP is observed, showing thus, there is no 315 predisposition of inclusion of CDP in the cavity of the sterically hindered β-CD”. In my opinion, to improve the quality of the manuscript and prove the formation of the inclusion complex, the author should perform additional experiments, for instance, 2D NMR, DSC, or IM-MS. The authors do not know if described interaction is internal or external. The cyclodextrin and chloroquine diphosphate create the external interaction, as the author mentioned: “However, interactions 44 between guest molecules and cyclodextrins can also occur via, for example, H-bonding 45 involving hydroxyl groups located outside the CD’s cavity [10,11]. This type of interaction does not improve drug stability.
The quality of the Scheme 1 should be improved.
The author's response to reviewers is as follows: This is a good point; interdiffusion coefficients reported here correspond to themaximum value of diffusion (or rate constant, if we assume a Fickian drug deliveryprocess –see, for example, J. Crank. The Mathematics of Diffusion) in a drug delivery process. Consequently, it can (or it must) be seen as a reference value. Unfortunately, it is not common to obtain this value from the literature due to the very strict experimental conditions required to measure this physical property. On the other hand, viscosity (in this case Newtonian viscosity) is quite relevant for different kind of formulations and also to understand if changes in diffusion coefficients results from alteration in the viscosity of solution or due to modification of the structure of the solution. At the end of the section 1, the following statement has been added: “Additionally, interdiffusion coefficients correspond to the maximum limit value for the release kinetics of drugs (or complexes), in drug delivery systems.” I agree with the authors if they prove the formation of the inclusion complex.

Comments for author File: Comments.pdf

Author Response

Reviewer #2

The authors added the new sentence: “Recently, it has been demonstrated by NMR and computational studies that chloroquine is able to protrude both α- and β–cyclodextrins, being that the stronger interaction occurs with the β-cyclodextrin [14].” the NMR data, only the computational studies. I had to find the extra references in the cited article that contained the information about NMR data. However, Roy et al. performed only the 1H NMR spectrum. They claimed that the shifting of ring protons is enough to prove the formation of the inclusion complex. The 1H NMR spectrum can only provide information about the creation of some interactions. The 1H NMR spectrum does not provide information about the types of interaction. I am not convinced that the highly hydrophobic chloroquine diphosphate possesses the ability to the formation of inclusion complex with a hydrophobic cyclodextrin cavity. Additionally, authors claimed that “For more concentrated solutions, for 314 which it is obtained that D12>0, a co-flow of CDP is observed, showing thus, there is no 315 predisposition of inclusion of CDP in the cavity of the sterically hindered β-CD”.
In my opinion, to improve the quality of the manuscript and prove the formation of the inclusion complex, the author should perform additional experiments, for instance, 2D NMR, DSC, or IM-MS. The authors do not know if described interaction is internal or external. The cyclodextrin and chloroquine diphosphate create the external interaction, as the author mentioned: “However, interactions 44 between guest molecules and cyclodextrins can also occur via, for example, H-bonding 45 involving hydroxyl groups located outside the CD’s cavity [10,11]. This type of interaction does not improve drug stability.

We are completely in agreement with the referee when he said that he is not convinced that the highly hydrophilic chloroquine diphosphate possesses the ability to the formation of inclusion complex with a hydrophobic cyclodextrin cavity. This is a very weak interaction between these solutes (which is supported by the small value that can be estimated for the equilibrium constant of the complexation between both solutes, CDP and β-CD, K = (30 ± 0.8) mol^-¹ dm³). In addition, as a result of the “salting-out effect”, β-CD concentration gradients can drive significant coupled flows of this drug. a co-flow of CDP is observed, showing thus, there is no predisposition of inclusion of CDP in the cavity of the sterically hindered β-CD. This result is consistent with weak chloroquine binding to cyclodextrin, in contrast to a recent report of anomalously large–chloroquine binding constants.

Besides, we have modified the text in order to delete the word “inclusion”.

Consequently, we have modified the conclusions.

The quality of the Scheme 1 should be improved.

Bearing in mind that the scheme is not in fact very enlightening, and considering that from our data, it was obtained a very low inclusion complex formation constant, the scheme was skipped.

The author's response to reviewers is as follows: This is a good point; interdiffusion coefficients reported here correspond to themaximum value of diffusion (or rate constant, if we assume a Fickian drug deliveryprocess –see, for example, J. Crank. The Mathematics of Diffusion) in a drug delivery process. Consequently, it can (or it must) be seen as a reference value. Unfortunately, it is not common to obtain this value from the literature due to the very strict experimental conditions required to measure this physical property. On the other hand, viscosity (in this case Newtonian viscosity) is quite relevant for different kind of formulations and also to understand if changes in diffusion coefficients results from alteration in the viscosity of solution or due to modification of the structure of the solution. At the end of the section 1, the following statement has been added: “Additionally, interdiffusion coefficients correspond to the maximum limit value for the release kinetics of drugs (or complexes), in drug delivery systems.” I agree with the authors if they prove the formation of the inclusion complex.

The answer to this question has already been given above.

Author Response File: Author Response.pdf