Review Reports - Saturated Micellar Networks: Phase Separation and Nanoemulsification Capacity

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This paper is well written and the experimental procedure and findings are easy to follow.

However I do not think this is exceptionally interesting. Why re the results applicable and why would anyone be interested in them?

As a theoretician I would also like to know hat goes on in the nanoscale. Why do different viscosities and turbidites and emulsions occur with different salts, their concentration and different surfactants.

Here are a few minor points:

1) In abstract define your acronyms. The abstract has to stand out on its own. Define acronyms in both the abstract and first mention in main text.

2) Lines 34-35. Refer to the molecules and surfacyants illustrated in Figure 1.

3) What is CAPB acronym?

4) Lines 105-116. Label and refernce names in the figure 1.

Author Response

Response to Reviewer #1

We thank Reviewer #1 for the positive assessment of our paper, for his/her comments and suggestions that helped us to improve the quality of the manuscript.

An itemized response to the points raised by the reviewer is following.

Reviewer #1 writes: “In abstract define your acronyms. The abstract has to stand out on its own. Define acronyms in both the abstract and first mention in main text.”

Response: Corrected.

Reviewer #1 writes: “Lines 34-35. Refer to the molecules and surfactants illustrated in Figure 1.”

Response: Corrected.

Reviewer #1 writes: “What is CAPB acronym?”

Response: Corrected.

Reviewer #1 writes: “Lines 105-116. Label and reference names in Figure 1.”

Response: Corrected.

General comments and questions:

Reviewer #1 writes: “Why are the results applicable and why would anyone be interested in them?”

In the Introduction, we included the following:

“When put in a contact with small fragrance molecules (limonene, linalool, citronellol and benzyl salicylate), the separated micellar phase engulfs easily the oils and upon dilution forms nanoemulsions with a droplet size around 130 nm. As a result, the energy needed for the formation of nanoemulsions upon dilution of the solubilized oils in the multiconnected network is considerably less in comparison with the conventional methods that used a mechanical drop breakup. The small droplet size implies that the saturated micellar networks can find an application in pharmaceutics as vehicles for drug delivery [24-30].”

In practice (detergents, personal care, cosmetics, drug delivery systems, etc.), the mixed micellar systems with a preliminary define composition (e.g. SLES-1EO+CAPB, SDS+DDAO, etc.) are widely used. In order to produce the final formulations, small amounts of fragrances, oils, Vitamins, etc. must be included in the form of nanoemulsion droplets. The general problem is that the conventional methods for a nanoemulsification (usage of all types of homogenizers) are with an extremely high energy consumption. In our case, the mixtures of saturated micellar networks and respective fragrances or Vitamin E form spontaneously nanoemulsion upon dilution. That is without any additional energy consumption. We believe that this approach will be of great interest for the chemical technology.

Reviewer #1 writes: “Why do different viscosities and turbidites and emulsions occur with different salts, their concentration and different surfactants?”

From the viewpoint of theoretical interpretation, the following models are known in the literature:

The growth of rodlike and wormlike micelles is modeled in the framework of the molecular thermodynamics for:
a) Nonionic surfactant micelles – Adv. Colloid Interface Sci. 256 (2018) 1–22. In this publication except of the theoretical approach, one can find a complete review on the state of art;
b) Mixed micelles of nonionic surfactants – J. Colloid Interface Sci. 547 (2019) 245–255 and 551 (2019) 227–241;
c) Mixed micelles of ionic and nonionic surfactants – J. Colloid Interface Sci. 581 (2021) 262–275 and 584 (2021) 561–581.
d) Zwitterionic surfactant micelles – J. Colloid Interface Sci. 627 (2022) 469–482.

These theoretical models explain the effects of the length of hydrocarbon tails, size and type of hydrophilic heads, effect of type and concentration of counterions (salt), surfactant concentration and ionic strength, etc. on rodlike and wormlike micelles. Unfortunately, these quantitative models are operative up to the formation of branched micelles. The formation of branched micelles is studied by qualitative molecular dynamics computer simulations. From our knowledge, theoretical predictions on the onset of saturated micellar networks and/or bicontinuous micellar phases are not discussed in the literature. As we showed [23,52] and in the present manuscript, the formation of these phases is a fine balance of surfactant and divalent salt concentrations.

The relationships between length and type of micelles and rheology, e.g. “salt curves” for the viscosity vs salt concentration. Note, that the available theories predict the bulk rheological behavior of micellar solutions, if one knows the shape and size of the micelles.

In this case, the most popular is the Cates theory known as “the reptation-reaction model”. This model explains the rise of the viscosity with the increase of the mean micellar length in the case of wormlike micelles and the decrease of the viscosity with the rise of the branching points for branched micelles. In this theoretical field, the most important Cates et al. publications are:

Cates M.E., Reptation of living polymers: dynamics of entangled polymers in the presence of reversible chain-scission reactions, Macromolecules 20 (1987) 2289–2296.

Cates M.E., Dynamics of living polymers and flexible surfactant micelles: scaling laws for dilution, J. Phys. E 49 (1988) 1593–1600.

Cates M.E., Nonlinear viscoelasticity of wormlike micelles (and other reversible breakable polymers), J. Phys. Chem. 94 (1990) 371–375.

Cates M.E., Candau S.J., Statistics and dynamics of worm-like surfactant micelles, J. Phys. Condens. Matter 2 (1990) 6869–6892.

Turner M.S., Cates M.E., Linear viscoelasticity of living polymers: a quantitative probe of chemical relaxation times, Langmuir 7 (1991) 1590–1594.

Kern F., Lemarechal P., Candau S.J., Cates M.E., Rheological properties of semidilute and concentrated solutions of cetyltrimethylammonium bromide in the presence of potassium bromide, Langmuir 8 (1992) 437–440.

Cates M.E., Fielding S.M., Rheology of giant micelles, Adv. Phys. 55 (2006) 799–879.

As it is seen, there are no published theoretical models related to the saturated micellar networks (from our knowledge), so that there are no discussions on the state of art in this field included in the manuscript.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

In this work, the authors described the phase properties of the mixture of anionic surfactants and zwitterionic surfactants, with the presence of divalent or monovalent ions. Specifically, the impact of ethylene oxide units in the anionic surfactant was illustrated. The rheological study revealed the phase transition under different salt concentrations. Additionally, oily substances (limonene and Vitamin E) were loaded to the SLES-1EO+DDAO and SLES-1EO+CAPB systems to their nanoemulsification capability. Many experiments were designed and conducted, however, the data analysis was not in-depth, focused, or conclusive enough in many cases. With many details provided in the text, certain study interest was not highlighted clearly. Also, several Figures didn’t match the description in the manuscript, probably due to the uncorrected change in Figure order. Therefore, this manuscript needs major revision and reorganization before its consideration for acceptance. Detailed comments are listed as follows:

1. On pages 4-5, section 3.1, the visual appearance of the mixture of SDS/SLES-1EO/SLES-3EO+DDAO was exhibited, along with the mixture of SDS/ SLES-3EO+CPAB but not SLES-1EO+CPAB. Only references were cited regarding SLES-1EO+CPAB. It’s important to illustrate the reported observation of SLES-1EO+CPAB under different MgCl2 and CaCl2 concentrations, facilitating readers to make a comparison with other systems described here. Additionally, it is critical to discuss the physicochemical reasons behind the impact of ethylene oxide units and the nature of zwitterionic surfactant on the phase diagram, e.g., hydrophobicity/hydrophobicity of the molecules, charge density, certain chemical groups, etc.

2. It is encouraged to generate clear phase diagrams on the discussed surfactant mixture systems, combining the results from visual observations and rheological analysis, to clearly summarize their phase properties under different salt concentrations. For example, to the system SLES-1EO+DDAO, its saturated micellar phases were illustrated to be with the presence of 50-70mM MgCl2 according to the photo in Fig. 2, while the rheological analysis in Fig. 6 further revealed the maximum MgCl2 being 20 mM before the phase separation starts. The differences in critical points need to be compared, discussed, and summarized for ease of understanding and following.

3. In section 4, page 12, the mixture systems SLES-1EO+DDAO and SLES-1EO+CAPB responded differently to the addition of Vitamin E. Any discussion on the potential physicochemical reason? Is this a specific case, or any general rule can be indicated? Similarly, any explanation for the higher loading capacity of limonene from SLES-1EO+CAPB (page 11)?

4. In Figures 8 and 9, the sizes of the saturated micellar phases were measured. How’s the stability of these saturated micellar phases? Is there a continuous phase separation or change in branched micelle size over time?

5. To the research in section 4, what is the motivation for loading the oily substances in the branched/saturated micellar phases? What is its advantage as compared to loading them in the micellar phases (under low salt conditions)? Additionally, on page 7 line 278, it was described that the monovalent salt can promote the formation of bicontinuous micellar phase, then why monovalent salt was not applied for the nanoemulsification of limonene and Vitamin E?

6. Several Figures didn’t match the description in the manuscript, probably due to the uncorrected change in Figure order: in line 299, Figure 7b should be Figure 6b instead; in line 305, Figure 7a and Figure 7c should be Figure 6a and Figure 6c instead; in line 322, Figure 8a should be Figure 7a instead.

Author Response

Response to Reviewer #2

We thank Reviewer #2 for the positive assessment of our paper, for his/her comments and suggestions that helped us to improve the quality of the manuscript.

An itemized response to the points raised by the reviewer is following.

Reviewer #2 writes: “On pages 4-5, section 3.1, the visual appearance of the mixture of SDS/SLES-1EO/SLES-3EO+DDAO was exhibited, along with the mixture of SDS/SLES-3EO+CPAB but not SLES-1EO+CPAB. Only references were cited regarding SLES-1EO+CPAB. It’s important to illustrate the reported observation of SLES-1EO+CPAB under different MgCl2 and CaCl2 concentrations, facilitating readers to make a comparison with other systems described here. Additionally, it is critical to discuss the physicochemical reasons behind the impact of ethylene oxide units and the nature of zwitterionic surfactant on the phase diagram, e.g., hydrophobicity/hydrophobicity of the molecules, charge density, certain chemical groups, etc.”

Response:

a) We added Figure 2c and Figure 2d to show the visual appearance of SLES-1EO+CAPB systems in the presence of divalent salts:

“Figures 2c and 2d show the visual appearance of 8 wt% SLES-1EO+CAPB solutions in the presence of different amount of MgCl₂ and CaCl₂. The behavior of the solutions is similar to that illustrated in Figures 2a and 2b. The main difference is in the intervals of concentrations in which saturated micellar phases are formed: i) from 60 mM to 160 mM for added MgCl₂; ii) from 50 mM to 70 mM for added CaCl₂. Thus, the type of zwitterion is also important for the formation of the sponge phases, see Section 3.2.”

b) We added Table S1 in the supplementary material, which summarizes the experimental data for the turbidity of 4 wt% of all studied surfactants in the presence of MgCl₂ and CaCl₂. It is shown that all solutions are transparent except of 4 wt% SDS in the presence of both MgCl₂ and CaCl₂ in the relatively low divalent salt concentrations (50-70 mM).
c) We summarized our phase diagrams in Table S2 for easier explanation.
d) With respect to the importance of the number of ethylene oxide groups, we added the following explanation:

“If the concentration of individual surfactant solutions is 4 wt%, then the addition of MgCl₂ or CaCl₂ up to 120 mM does not change the turbidity of all surfactant solutions (they are transparent) except of SDS (see Table S1). In the case of SDS, the crystallites are observed for concentrations above 50 mM added MgCl₂ and even at 50 mM added CaCl₂. It is well-known that the counterion binding energy (Mg²⁺ and Ca²⁺) to the ionic surfactant headgroups decreases with the rise of the number of ethylene oxide groups. For that reason, the lowest counterion binding energy to SLES-3EO leads to the growth of wormlike mixed micelles and to the monotonical increase of the viscosity of solutions up to 350 mM added MgCl₂ in both cases of SLES-3EO+ DDAO and SLES-3EO+ CAPB. The highest energy of counterion binding to SDS leads to the salting out of surfactants for low concentrations of added divalent salts. The following general conclusions can be drawn. The increase of the number of ethylene oxide groups in ionic surfactants prevents the transition of the branched micelles to a multi-connected micellar phase in the presence of divalent salts.”

With respect to the effect of the type of zwitterions we added the following explanation:

“The reported differences between DDAO and CAPB containing solutions needs a deeper explanation. There are three main differences between used zwitterionic surfactants. First, the CAPB samples contain NaCl while DDAO sample is salt free (see Section 2). To clarify the effect of NaCl available in the CAPB sample, we prepared mixtures of DDAO and NaCl (100 mM CAPB contains 118 mM NaCl) which mimic the used CAPB sample. The whole experimental procedure was repeated using these mixtures of 8 wt% 1:1 SLES-1EO+DDAO and 138 mM NaCl instead of DDAO alone. In the presence of NaCl, the saturated micellar phases are obtained in the case of added 50, 60, and 70 mM MgCl2 (see Figure S8a) – exactly the same interval of MgCl₂ concentrations illustrated in Figure 2a. Thus, this admixture of NaCl (138 mM) does not affect the formation of the respective bicontinuous micellar phases. Nevertheless, the monovalent salt affects the rheological behavior of the transparent micellar solutions, see Figures 7b and S8b. Wormlike micellar solutions with approximately equal viscosities of about 30 Pa.s are observed at concentrations of added MgCl₂ below 10 mM and even without added MgCl₂. The subsequent increase of divalent salt concentration leads to the formation of branched micelles and to the decrease of the viscosity. Thus, the dependence of the viscosity vs MgCl₂ concentration has not a typical shape of the salt curves. All experimental results show that the energy of counterion binding of Mg²⁺ and Ca²⁺ to the micelles is larger than that of Na⁺ ions. For that reason, 50 mM Mg²⁺ ions added in the solution are capable to displace bound Na⁺ ions to micelles (138 mM added bulk concentration of NaCl + 142 mM Na⁺ ions from the dissociation of SLES-1EO = 280 mM Na⁺ ions). Nevertheless, the zero-shear viscosities again are lower than the maximum of h_app shown in Figure 7a for CAPB containing solutions.

Second, the composition of CAPB is different – it is a mixture of zwitterions with a various number of carbon atoms in the hydrophobic chains (from C8 to C16, in which 48% are C12), while DDAO is a zwitterion with 12 carbon atoms in the hydrophobic chain. The longer lengths of the CAPB hydrophobic chains lead to the easier formation of wormlike micelles and promote the micelle growth with the rise of added salt concentration. This is the most probable explanation of the higher viscosities of the CAPB containing solutions.

Third, the dipole length of CAPB is higher than that of DDAO (Figure 1). In the micelles, the negative surfactant head of SDS is close to the positive charge of the DDAO and CAPB dipole heads. Because of the higher dipole length of CAPB, it shields the charge of SDS molecule in the mixed micelles more effectively than that of DDAO. As a result, SDS+CAPB can form saturated micellar phase, while SDS+DDAO solutions precipitates for concentration of added divalent salts above 25 mM (see Table S2).”

Reviewer #2 writes: “It is encouraged to generate clear phase diagrams on the discussed surfactant mixture systems, combining the results from visual observations and rheological analysis, to clearly summarize their phase properties under different salt concentrations. For example, to the system SLES-1EO+DDAO, its saturated micellar phases were illustrated to be with the presence of 50-70 mM MgCl2 according to the photo in Fig. 2, while the rheological analysis in Fig. 6 further revealed the maximum MgCl2 being 20 mM before the phase separation starts. The differences in critical points need to be compared, discussed, and summarized for ease of understanding and following.”

Response: See point 1.

Reviewer #2 writes: “In section 4, page 12, the mixture systems SLES-1EO+DDAO and SLES-1EO+CAPB responded differently to the addition of Vitamin E. Any discussion on the potential physicochemical reason? Is this a specific case, or any general rule can be indicated? Similarly, any explanation for the higher loading capacity of limonene from SLES-1EO+CAPB (page 11)?”

Response: We included the following explanation in the revised version:

“The nanoemulsification capacity of SLES-1EO+CAPB saturated micellar phases decreases with the rise of the van der Waals volume of the oil molecule and does not correlate with its lipophilicity characterized by logP [23]. In our case, the van der Waals volume of limonene is 154.7 Å³ and that of Vitamin E is in the order of 430 Å³ [59]. This explains the lower nanoemulsification capacity observed in the case of Vitamin E.”

Reviewer #2 writes: “In Figures 8 and 9, the sizes of the saturated micellar phases were measured. How’s the stability of these saturated micellar phases? Is there a continuous phase separation or change in branched micelle size over time?”

Response: We added the following explanation in the revised version of the manuscript:

“It is important to note, that all isolated saturated micellar phases and the respective obtained nanoemulsions (Figures 8-10) are thermodynamically stable. There are no phase separation or oil lenses seen and no change of the size distributions at least for three months.”

Reviewer #2 writes: “To the research in section 4, what is the motivation for loading the oily substances in the branched/saturated micellar phases? What is its advantage as compared to loading them in the micellar phases (under low salt conditions)? Additionally, on page 7 line 278, it was described that the monovalent salt can promote the formation of bicontinuous micellar phase, then why monovalent salt was not applied for the nanoemulsification of limonene and Vitamin E?”

Response: In fact, the main practical question is the following. If one has branched micellar solutions without added salt and the isolated saturated micellar phase containing small amount of divalent salt with the same surfactant concentrations, then one can add equal amount of e.g. limonene to both solutions. This is shown in Figure 8a and Figure 8b. Upon dilution of these solutions, one obtains swollen micelles (from branched micellar solutions) and nanoemulsions (from saturated micellar phases), see Figure 8c and Figure 8d. As a result, the branched micellar solutions cannot be used for nanoemulsion preparation, while the saturated micellar networks spontaneously form nanoemulsions upon dilution. The nanoemulsions with fragrances and Vitamins are those from a practical interest in cosmetics, personal care products, drug delivery systems, etc. mentioned in the Introduction.

Second, the nanoemulsification capacity of the system with high salinity (with added NaCl) is not included in the manuscript because they have not practical application (from our knowledge).

Reviewer #2 writes: “Several Figures didn’t match the description in the manuscript, probably due to the uncorrected change in Figure order: in line 299, Figure 7b should be Figure 6b instead; in line 305, Figure 7a and Figure 7c should be Figure 6a and Figure 6c instead; in line 322, Figure 8a should be Figure 7a instead.”

Response: Corrected.

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript deals with the investigation of phase separation and nano-emulsification ability of surfactants mixtures (anionic + switterionic) able to form saturated micellar networks in the presence of salts. The manuscript requires substantial revisions.

Introduction: The introduction is not well organised and it should be rewritten or strongly modified. The subject related to the topic of saturated micellar network (or phases) is not well introduced. Moreover, the introduction should firstly introduce well the general topic of the manuscript (saturated micellar phases, in this cases), then focus on the specific question, that the experiments performed have addressed, report the related scientific background, and finally state the aim of the study.

Materials: Line 111-112 The sentence is not clear. Does it mean that CAPB is not pure? The purity of all surfactants should be carefully reported.

Methods: Methods are not sufficiently described. For instance, the concentration of surfactants are not reported. The details about SAXS measurements are not reported. From the figure 6B in the results section it seems that the maximum shear rate applied was 200 s^-1 and not 1000 s^-1. Some procedures explained in the results section are not detailed in the method section. For instance, as the saturated micellar phases were separated from the samples. Method for DLS is incomplete and erroneous. It is not reported the operating temperature and, in the case of viscous samples as for the saturated micellar phases, the measurement cannot be reliable since particles do not move according to Brownian motion. DLS measured the scattered light intensity at a fixed angle (90° or 173°) and not over a wide range of scattering angles as reported in the manuscript.

Results:

Line 212 Why micelles are referred as droplets? Should they be particles? Why do they appear as droplets under microscopic observation (Figure 4C and Figure 4D)?

Line 226-230 The sentence should be modified since interfacial tension and elasticity were not measured.

Line 248-250 Please explain better

Line 252-264 it is not clear how the authors demonstrated the presence of bicontinuous structures.

Line 256 I think that the term supernatant here is not correctly used since it is lower phase. I think it would be better to replace supernatant with lower phase or bottom phase.

“Thus, the upper phase contains enough water molecules” the sentence is not clear.

Line 260 Polarised optical microscope is not mentioned in the method and the results are not presented, therefore the lack of birefringence cannot be stated.

Line 260 How SAXS diffraction spectrum “proves that the upper phase represents a multi-connected micellar network? SAXS experiments should be better described in the method and results achieved in the main text. Figure S4d could be moved into the main text, if adequately discussed.

Paragraph 3.2 Rheological analyses should be better described.

Line 295-296 How branching of micelles and increase of junction can decrease the measured viscosity?

Line 298 How the bicontinuous phase was isolated?

Lie 304-305 The rheological behaviour of the saturated micellar phase should be better described. Here, it is referred as Newtonian or quasi-Newtonian, but according to Figure 6B, the measured viscosity is not constant and independent from the applied shear rate. Moreover, rheological profiles in the supplementary materials are different.

Figure 7 can be moved to the supplementary materials since it is redundant.

Line 322 Figure 8a is erroneously cited

Line 323-333 This part is confusing about comparisons. Moreover, the discussion is mainly based on the Newtonian viscosity, but it has never defined before, and eventually, it has not reported as it has been calculated from the experimental data.

Line 339 What does it mean “the solid lines are guide to the eye”?

Line 361-365 The operating procedure is not clear.

Line 371 It should be further confirmed that Limonene is encapsulated into micelles. Moreover, how limonene was added is not reported.

Paragraph 4. I really think that the only DLS experiments performed at these conditions cannot demonstrate that limonene and vitamin D can be nanoemulsified by the saturated micellar phase. Moreover, the nano-emulsification may occur only after dilution and the authors did not investigate at which conditions. In which state is limonene and vitamin D in the saturated micellar phase before diluting? The authors should at least provide optical microscope images of saturated micellar phase loaded with limonene and vitamin D both before and after dilution, and they should discuss them. As said before, DLS results could not be reliable. Please consider also whether is appropriate modified the title in relation to the nano-emulsification ability

Figure 9 can be moved to supplementary materials.

Conclusions and abstract must be revised accordingly to the modification in the text.

Line 485-486 Elasticity, yield stress and interfacial tension have not been measured.

Line 487-489 Please modify

Line 495 10% w/w limonene was not tested since only 1% and 8% was tested according to the results section.

Comments on the Quality of English Language

Some sentences can be improved.

Author Response

Response to Reviewer #3

We thank Reviewer #3 for the positive assessment of our paper, a constructive criticism, his/her comments and suggestions that helped us to improve the quality of the manuscript.

An itemized response to the points raised by the reviewer is following.

Reviewer #3 writes: “Introduction: The introduction is not well organized and it should be rewritten or strongly modified. The subject related to the topic of saturated micellar network (or phases) is not well introduced. Moreover, the introduction should firstly introduce well the general topic of the manuscript (saturated micellar phases, in this cases), then focus on the specific question, that the experiments performed have addressed, report the related scientific background, and finally state the aim of the study.”

Response: The introduction is rewritten following exactly your suggestions.

Reviewer #3 writes: “Materials: Line 111-112 The sentence is not clear. Does it mean that CAPB is not pure? The purity of all surfactants should be carefully reported.”

Response: The purity of used surfactants is reported in the revised version. In the case of CAPB we added the following explanation:

“The used CAPB sample is a mixture of zwitterions with a different number of carbon atoms in the hydrocarbon chains (from C8 up to C16, of which 48% is C12) and NaCl (not specified by the manufacturer). By conductometry, we established that 100 mM of the used CAPB contains an admixture of 118 mM NaCl [5].”

Reviewer #3 writes: “Methods: Methods are not sufficiently described. For instance, the concentration of surfactants are not reported. The details about SAXS measurements are not reported. From the figure 6B in the results section it seems that the maximum shear rate applied was 200 s-1 and not 1000 s-1. Some procedures explained in the results section are not detailed in the method section. For instance, as the saturated micellar phases were separated from the samples. Method for DLS is incomplete and erroneous. It is not reported the operating temperature and, in the case of viscous samples as for the saturated micellar phases, the measurement cannot be reliable since particles do not move according to Brownian motion. DLS measured the scattered light intensity at a fixed angle (90° or 173°) and not over a wide range of scattering angles as reported in the manuscript.”

Response: The concentration of surfactants is shown in Section 2.2. The operating temperature of 25 ^oC and the procedure for the isolation of the saturated micellar phases are added in the revised manuscript – see point 14 below. In rheological experiments, the applied shear rates are from 0.001 s^-¹ to 1000 s^-¹ (see Figures S5–S8). For better illustration, the shear rates in Figure 6b are shown to 200 s^-¹.

The following explanation on SAXS measurements is added:

“To confirm the optical observations, we used also small-angle X-ray scattering (SAXS) system Xeuss 3.0 (SAXS/WAXS System, Xenocs) with a CuKa X-ray source (λ = 0.154 nm, Xeuss 3.0 UHR Dual source Mo/Cu, Xenocs, Sassenage, France) and Eiger2 4 M detector (Dectris Ltd., Baden Deattwil, Switzerland) with slit collimation. The apparatus was operated at 50 kV and 0.6 mA. The sample to detector distance (SDD) of 1000 mm and 3000 mm allowed to access the Q-range of 0.01–0.5 Å⁻¹. Data acquisition time was 30 min. Samples were enclosed into vacuum tight thin borosilicate capillary with an outer diameter of 1 mm and thickness of 10 µm. The scattered intensity was normalized to the incident intensity, and were corrected for the background scattering from the capillary. It was calibrated to absolute scale. The measurements were performed at 25 °C.”

In the case of DLS we modified the sentence as follows:

“For measuring the size distribution of emulsion drops in the nanoemulsions, we used dynamic light scattering (DLS) apparatus LS Spectrometer™ from LS Instruments AG. The angle of the measurements is fixed at 90° and the temperature is 25 °C.”

Note that we used DLS only to characterize the sizes of nanoemulsion droplets, not the structure of the saturated micellar network. There are no aggregates, particles, etc. in the obtained nanoemulsions and their viscosities are low.

Reviewer #3 writes: “Line 212 Why micelles are referred as droplets? Should they be particles? Why do they appear as droplets under microscopic observation (Figure 4C and Figure 4D)?”

Response: You are right. The term „droplets“ is not correct. The text is modified as follows:

“Note that 8 wt% 1:1 SLES-1EO+DDAO + 50 mM CaCl₂ solution is quite turbid. In this case, the saturated micellar phase appears as very small fluid aggregates, which do not coalescence to a large uniform layer on the top of the vial even after 24 hours.”

For better explanation we modified the respective sentences as follows:

“In transmitted light, one sees several fluid aggregates in the form of “droplets” dispersed in the continuous phase (no color is detected due to the fact that the hydrophobic dye is fluorescent). In fluorescence mode, one proves that the continuous phase (the background of the picture) is the saturated micellar phase (due to the intense green color from the hydrophobic dye dissolved in it), and the dark “droplets” are from the water-rich phase.”

Reviewer #3 writes: “Line 226-230 The sentence should be modified since interfacial tension and elasticity were not measured.”

Response: We modified the respective sentences as follows:

“We tried several times to suck out the lower phase with syringe and we observed that the needle cannot go through the boundary between the two phases – instead of penetrating, the needle bends the interface and easily deforms the boundary between phases (Figure 3d). Thus, the interface behaves as an elastic membrane which is very flexible to bending (low interfacial tension).”

Moreover, we tried to measure the interfacial tension by the drop shape analysis. Unfortunately, it was impossible to make a drop from the isolated saturated micellar phase attached to the capillary in a water-rich phase – instead of a drop, the continuous jet is produced. The same problem we had using the spinning drop method. This behavior is typical for interfaces with very low interfacial tension. The jets from the saturated phase bend and flow in the water-rich phase without mixing.

Reviewer #3 writes: “Line 248-250 Please explain better.”

Response: Corrected.

Reviewer #3 writes: “Line 252-264 it is not clear how the authors demonstrated the presence of bicontinuous structures.”

Response: See the comments included in point 4.

Reviewer #3 writes: “Line 256 I think that the term supernatant here is not correctly used since it is lower phase. I think it would be better to replace supernatant with lower phase or bottom phase.”

Response: Corrected.

Reviewer #3 writes: ““Thus, the upper phase contains enough water molecules” the sentence is not clear.”

Response: Corrected.

Reviewer #3 writes: “Line 260 Polarised optical microscope is not mentioned in the method and the results are not presented, therefore the lack of birefringence cannot be stated.”

Response: Corrected. We added Figure S10 and the explanation in Section 2.

“Some of the experiments were performed in а cross-polarized white light with l-compensator plate placed after the observed sample and before the analyzer at 45° angle with respect to both polarizer and analyzer.”

Reviewer #3 writes: “Line 260 How SAXS diffraction spectrum “proves that the upper phase represents a multi-connected micellar network? SAXS experiments should be better described in the method and results achieved in the main text. Figure S4d could be moved into the main text, if adequately discussed.”

Response: Corrected.

Reviewer #3 writes: “Paragraph 3.2 Rheological analyses should be better described.”

Response: Corrected. See point 13, 15, etc.

Reviewer #3 writes: “Line 295-296 How branching of micelles and increase of junction can decrease the measured viscosity?”

Response: We included in the main text:

“The Cates theory [5-13] known as “the reptation-reaction model” for living polymers describes the relationships between the size, shape, and self-organization of micelles and the rheological behavior of the respective solutions. For example, when branched micelles are formed, the endcaps of the wormlike micelles are transformed into junctions in a branched structure. These junctions are mobile and they can slide along the linear part of the micelles, thus decreasing the viscosity of the surfactant solutions.”

Reviewer #3 writes: “Line 298 How the bicontinuous phase was isolated?”

Response: In Section 2.2 we added the following explanation:

“The solutions are left overnight in a separation funnel to separate the saturated micelle network (if any). After that we carefully pour the lower phase in a beaker, the upper phase transferred in another beaker, thus separating both phases. All subsequent measurements are carried at 25 ^oC.”

Reviewer #3 writes: “Line 304-305 The rheological behaviour of the saturated micellar phase should be better described. Here, it is referred as Newtonian or quasi-Newtonian, but according to Figure 6B, the measured viscosity is not constant and independent from the applied shear rate. Moreover, rheological profiles in the supplementary materials are different.”

Response:

“The original experimental flow curves (apparent viscosities vs the applied shear rate, dg/dt) are summarized in Figures S5 and S6. It is well illustrated the typical rheological behavior of the studied solutions for concentrations below the phase separation. At low shear rates, the apparent viscosity is constant (quasi-Newtonian regime). These values of the viscosity are called in the literature the zero-shear viscosities and they are plotted in the “salt curves” illustrated in Figures 6 and 7. The quasi-Newtonian regime takes place to a threshold shear rates and the subsequent increase of dg/dt leads to the shear thinning behavior – the apparent viscosity decreases with dg/dt. At high enough shear rates, the apparent viscosity again is constant, which corresponds to Newtonian fluids. Note that for SLES-1EO+DDAO in the presence of low concentrations of divalent salts, the apparent viscosity is constant for the all studied shear rates, so that these solutions behave as Newtonian fluids.”

Reviewer #3 writes: “Figure 7 can be moved to the supplementary materials since it is redundant.”

Response: According to Reviewer 2, Figure 7 is very important to clarify the effect of NaCl appearing in the CAPB sample compared to DDAO sample, which is salt free.

Reviewer #3 writes: “Line 322 Figure 8a is erroneously cited”

Response: Corrected.

Reviewer #3 writes: “Line 323-333 This part is confusing about comparisons. Moreover, the discussion is mainly based on the Newtonian viscosity, but it has never defined before, and eventually, it has not reported as it has been calculated from the experimental data.”

Response: See point 15.

Reviewer #3 writes: “Line 339 What does it mean “the solid lines are guide to the eye”?”

Response: Corrected.

Reviewer #3 writes: “Line 361-365 The operating procedure is not clear.”

Response: The following explanation is added to the main text:

“To obtain the total surfactant concentration in the separated saturated micellar phase from 8 wt% 1:1 SLES-1EO+DDAO, we placed the sample in an oven at 50 °C and left it there for several days. During this period of time, the petri dish is regularly taken out of the oven and weighted until the weight of the petri dish + saturated micellar phase reached a constant value. From the weight of the initial (wet) sample of the saturated micellar phase and the respective final constant weight of the dried sample, we observed that the total surfactant concentration is 12 wt% (442 mM).”

Reviewer #3 writes: “Line 371 It should be further confirmed that Limonene is encapsulated into micelles. Moreover, how limonene was added is not reported.”

Response: The procedure for the limonene addition to the micellar solutions was described in details (see the second paragraph in Section 4). The fact that limonene is solubilized into micelles was confirmed by the measured larger mean micellar sizes in the presence of limonene (5.5 nm) compared to those without added limonene (4.8 nm).

Reviewer #3 writes: “Paragraph 4. I really think that the only DLS experiments performed at these conditions cannot demonstrate that limonene and vitamin D can be nanoemulsified by the saturated micellar phase. Moreover, the nano-emulsification may occur only after dilution and the authors did not investigate at which conditions. In which state is limonene and vitamin D in the saturated micellar phase before diluting? The authors should at least provide optical microscope images of saturated micellar phase loaded with limonene and vitamin D both before and after dilution, and they should discuss them. As said before, DLS results could not be reliable. Please consider also whether is appropriate modified the title in relation to the nano-emulsification ability.”

Response: The dimensions of the bicontinuous structure with limonene are in the nanoscale which makes observation with optical microscope impossible. We tried to make pictures but before dilution the background is homogeneous. One possibility is to use Cryo TEM in order to prove the type of the structures and the incorporation of limonene in the junctions of the micellar network.

Reviewer #3 writes: “Figure 9 can be moved to supplementary materials.”

Response: Figure 9 shows that the formed nanoemulsions after dilution have approximately equal size distribution, which is independent on the amount of limonene included in the saturated micellar phase (1 wt% or 7 wt%).

Reviewer #3 writes: “Conclusions and abstract must be revised accordingly to the modification in the text.”

Response: Corrected.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

This paper is hard to read for some one like me, who is not really an experimentalist. It would be easier to read if there were some discussion on the theoretical level. The authors did expand theoretical discussion though. Please explain things on further details about why such results occur on the molecular level.

Author Response

We thank Reviewer #1 for the positive assessment of our paper, for his/her comments and suggestions that helped us to improve the quality of the manuscript.

An itemized response to the points raised by the reviewer is following.

Reviewer #1 writes: “This paper is hard to read for some one like me, who is not really an experimentalist. It would be easier to read if there were some discussion on the theoretical level. The authors did expand theoretical discussion though. Please explain things on further details about why such results occur on the molecular level.”

Response: First, in the revised version of the manuscript, the following theoretical explanation of the rheological behavior was included according to the previous suggestions of Reviewer #2. We are sorry that we did not mention this in our previous reply to your comments. The following text appears in the revised version:

“The interpretation of rheological curves is well known in the literature [3-5, 8-13]: i) to the left of the maximum, the viscosity increases due to the growth and entanglement of the worm-like micelles; ii) to the right of the maximum, the viscosity decreases because of the branching of micelles and the increase of the junctions between them. The Cates theory [5-13] known as “the reptation-reaction model” for living polymers describes the relationships between the size, shape, and self-organization of micelles and the rheological behavior of the respective solutions. For example, when the branched micelles are formed, the endcaps of the wormlike micelles are transformed into junctions in the branched structure. These junctions are mobile and they can slide along the linear part of the micelles, thus decreasing the viscosity of the surfactant solutions.”

Second, according to your suggestion we added the following explanation in the second-round revision manuscript:

“The initial rise of the viscosity with salt concentration is related to the growth of the wormlike micelles in the surfactant solutions. In the framework of the molecular thermo-dynamics, the self-assembly of molecules in micelles depends on the total interaction free energy between them in the micellar phase. The following components of the total interaction energy are considered in the literature [58-63]: i) interfacial free energy due to the contact area of the micelle hydrocarbon core with outer aqueous phase; ii) chain conformation energy of molecules in the micellar phase; iii) headgroup steric repulsion energy; iv) electrostatic interaction component for ionic surfactant molecules; v) zwitterionic dipoles interaction component. The fine balance between different components of the interaction energy explains the effects of the length of hydrocarbon tails, size and type of hydrophilic heads, effect of type and concentration of counterions (salt), surfactant concentration and ionic strength, etc. on the molecular self-assembly in rodlike and wormlike micelles. The formation of branched micelles is studied by qualitative molecular dynamics computer simulations. From our knowledge, theoretical predictions on the onset of saturated micellar networks and/or bicontinuous micellar phases are not discussed in the literature.”

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The authors addressed the comments with adequate corrections and supplemented the manuscript with further discussions. This version of work has deeper insights and thus greatly improved the scientific soundness of the work.

There are some follow-up suggestions related to comment #5: the authors made the research motivation clear in the reply to comment #5, which is also recommended to be emphasized in section 4 (the practical need to form nanoemulsion upon dilution), not just in the introduction. Additionally, DLS results in Fig. 8c and 8d are measured after dilution, which should be mentioned in the figure caption. Similar to the figure captions of Fig. 9 and Fig. 10, if applicable.

The manuscript can be considered acceptable after the minor revision.

Author Response

We thank Reviewer #2 for the positive assessment of our paper, for his/her comments and suggestions that helped us to improve the quality of the manuscript.

An itemized response to the points raised by the reviewer is following.

Reviewer #2 writes: “The authors made the research motivation clear in the reply to comment #5, which is also recommended to be emphasized in section 4 (the practical need to form nanoemulsion upon dilution), not just in the introduction.”

Response: We added the following explanation in Section 4:

“The main practical question is the following. If one has branched micellar solutions without added salt and the isolated saturated micellar phase containing small amount of divalent salt with the same surfactant concentrations, then one can add equal weight frac-tions of fragrances and Vitamin E to both solutions. Upon dilution of these solutions, one obtains swollen micelles (from branched micellar solutions) and nanoemulsions (from saturated micellar phases), see Figure 8c and Figure 8d. As a result, the branched micellar solutions cannot be used for nanoemulsion preparation, while the saturated micellar networks spontaneously form nanoemulsions upon dilution. The nanoemulsions with fragrances and Vitamins are those from a practical interest in cosmetics, personal care products, drug delivery systems, etc. mentioned in the Introduction.”

Reviewer #2 writes: “Additionally, DLS results in Fig. 8c and 8d are measured after dilution, which should be mentioned in the figure caption. Similar to the figure captions of Fig. 9 and Fig. 10, if applicable.”

Response: Corrected.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

7. Reviewer #3 writes: “Line 252-264 it is not clear how the authors demonstrated the presence of bicontinuous structures.”

Response: See the comments included in point 4.

Reviewer answer: The authors should demonstrate better the presence of bicontinuous structure.

11. Reviewer #3 writes: “Line 260 How SAXS diffraction spectrum “proves that the upper phase represents a multi-connected micellar network? SAXS experiments should be better described in the method and results achieved in the main text. Figure S4d could be moved into the main text, if adequately discussed.”

Response: Corrected.

Reviewer answer: SAXS experiments should better described to assess the “multi-connected micellar structure” and to confirm the bicontinuous phase.

21. Reviewer #3 writes: “Line 371 It should be further confirmed that Limonene is encapsulated into micelles. Moreover, how limonene was added is not reported.”

Reviewer answer: The procedure for limonene addition should be reported in the method section and not results section. The presence of limonene should be further confirmed by an analytical determination into the isolated micellar phase. The slight increase in the particle size from DLS does not assess the encapsulation.

22.Reviewer #3 writes: “Paragraph 4. I really think that the only DLS experiments performed at these conditions cannot demonstrate that limonene and vitamin D can be nanoemulsified by the saturated micellar phase. Moreover, the nano-emulsification may occur only after dilution and the authors did not investigate at which conditions. In which state is limonene and vitamin D in the saturated micellar phase before diluting? The authors should at least provide optical microscope images of saturated micellar phase loaded with limonene and vitamin D both before and after dilution, and they should discuss them. As said before, DLS results could not be reliable. Please consider also whether is appropriate modified the title in relation to the nano-emulsification ability.”

The authors should be clearly state that the nanoemulsification occurs after diluting and they should also explain the reason of that. Actually, they presented a low-energy strategy for preparing nanoemulsions. I agree that nanoemulsions cannot be visualize by optical microscopy. Optical microscope image should be provided to assess that oil droplets at micrometric range do not form, thereby the emulsified system formed is a real nanoemulsions, with oil droplets only in the range of nanometers. The authors should explain better or provide references about the state of the compounds in the saturated micellar phase. How can they state that limonene and vitamin E are “homogeneously dispersed in the junctions of the micellar network” or “the limonene is dispersed into droplets and the solution is very turbid. The turbidity is due to the solubilized limonene in the junctions of the micellar network?

Author Response

We thank Reviewer #3 for the positive assessment of our paper, his/her comments and suggestions that helped us to clarify some points in the manuscript.

An itemized response to the points raised by the reviewer is following.

Reviewer #3 writes: “The authors should demonstrate better the presence of bicontinuous structure.”
Reviewer #3 writes: “SAXS experiments should better described to assess the “multi-connected micellar structure” and to confirm the bicontinuous phase.”

Response to points 1 and 2: We included the following explanation:

“To confirm the bicontinuous structure of the micellar network, we used BODIPY and water-soluble methylene blue dyes. The photograph of the vial containing 8 wt% 1:1 SLES-1EO+DDAO + 70 mM CaCl₂ shows that both dyes (lipophilic and hydrophilic) color the upper phase, which is in intense blue-green color (Figure S4a). The intensity of the blue color of the lower phase is low. Thus, the upper phase contains enough water in order to dissolve the methylene blue and to turn blue-green. After dispersing the aqueous phase in the dark blue phase, one sees again “droplets” in transmitted light (Figure S4b). In a fluorescence mode, the same “droplets” are dark, while the background in green (Figure S4c). This fact, combined with the lack of birefringence in polarized light (Figure S10) and the shape of the SAXS diffraction spectrum which does not correspond to any ordered liquid-crystal phase (Figure S4d), proves that the upper phase represents a multi-connected micellar network. Analogous SAXS diffraction spectrums are reported in the literature [53-57] and the authors attributed the respective shapes of the SAXS curves to the presence of saturated micellar (or sponge) phase. For example, in Ref. [53] the authors studied the sponge phase formation from 18 mM N-oleoyl b-_D-galactopyranosylamine and 13.5 mM octylphenoxypolyethoxyethanol aqueous solutions. They used lipophilic and hydrophilic dyes to characterize the visual appearance of analogous “droplets” containing sponge phase and SAXS measurements. The shape of the SAXS curves is similar to our observation shown in Figure S4d. From the position of the single broad peak (q_max = 0.12 Å^-¹), we estimated the characteristic length of the sponge phase (d = 2p/q_max = 5.2 nm). According to Ref. [53], this length can be interpreted as the average channel diameter of the sponge phase.”

Reviewer #3 writes: “The procedure for limonene addition should be reported in the method section and not results section. The presence of limonene should be further confirmed by an analytical determination into the isolated micellar phase. The slight increase in the particle size from DLS does not assess the encapsulation.”

Response: First, we moved the procedure for limonene and Vitamin E addition in the method section. The following explanation is added therein:

“To characterize the nanoemulsification capacity of the saturated micellar phases with respect to the added limonene and Vitamin E, we prepared large amounts of saturated micellar phases and left them to rest for the complete phase separation. The isolated phases are divided in equal portions and different concentrations of colored limonene or Vitamin E are added. The vials are stirred for 1 hour at room temperature and after that are left at least for one week at rest before the subsequent investigations.”

Second, the size of micelles without added limonene in the solution is 4.8 nm, which corresponds to spherical micelles [65]. In the case of added limonene, we measured size of micelles 5.7 nm. This size corresponds to swollen micelles because this size does not change at least for 3 month (mentioned in the man text of the manuscript) and the polydispersity is low. We have solubilization of limonene in the micelles not “encapsulation”. There is no reason to performed “analytical determination of limonene into micellar phase” because there is no phase separation – the limonene is homogenously dispersed in the saturated micellar phase.

Reviewer #3 writes: “The authors should be clearly state that the nanoemulsification occurs after diluting and they should also explain the reason of that. Actually, they presented a low-energy strategy for preparing nanoemulsions. I agree that nanoemulsions cannot be visualize by optical microscopy. Optical microscope image should be provided to assess that oil droplets at micrometric range do not form, thereby the emulsified system formed is a real nanoemulsions, with oil droplets only in the range of nanometers. The authors should explain better or provide references about the state of the compounds in the saturated micellar phase. How can they state that limonene and vitamin E are “homogeneously dispersed in the junctions of the micellar network” or “the limonene is dispersed into droplets and the solution is very turbid. The turbidity is due to the solubilized limonene in the junctions of the micellar network?”

Response: First, in all places of the main text and the abstract it is noticed that the nanoemulsions are obtained upon dilution of the dispersion from saturated micellar phase and limonene or Vitamin E. We also included Ref. [64] where it is shown that fragrances are solubilized in the junctions of the micellar network.

Author Response File: Author Response.pdf