Amphiphilic Diblock Copolymers of Poly(N-vinyl pyrrolidone) and Poly(vinyl esters) Bearing N-Alkyl Side Chains for the Encapsulation of Curcumin and Indomethacin

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This paper, by Plachouras et al., deals with the synthesis of amphiphilic diblock copolymers of poly(N-vinyl pyrrolidone) and poly(vinyl esters) bearing N-alkyl side chains by RAFT polymerization. Different copolymers with various poly(vinyl esters) (PVEs) blocks, molecular weights and compositions were synthesized. The self-assembly behavior of these copolymers in aqueous solution was then studied by DLS and SLS. Lastly, the encapsulation of hydrophobic curcumin and indomethacin within the core of the self-assemblies was studied in aqueous solution. Each step of the work is well detailed. The manuscript is well written and easy to read. However, some points need to be clarified. Therefore, I recommend its publication in Polymers after the following major revisions:

Even though the synthesis of the copolymers has already been published, it would have been good to remind the main synthesis steps in the SI and to show some data from the best systems in the SI, such as NMR spectra and GPC traces.

Page 4 Table 1: “seize” should be replaced by “size” in the caption below Table 1.

Page 4 line 136: Please precise how the water was added to the vial. Did you use a syringe pump? What was the flow rate?

Page 6 Table 2: What is the difference between R_h0, R_h1,0, and R_h2,0?

Page 7 line 248-249: How do you estimate the fraction of micellar aggregates and therefore their increase? Same question for the percentage ranges from 65% to 80% mentioned in line 257 for samples PNVP-b-PVBu #3, #4 and #5?

Besides, could you please precise at which concentration the DLS results presented in Table 2, Table 3, Table 4 were obtained?

Figure 1, Figure 3, Figure 4, and Figure 6 are not commented on.

For the DLS analyses, it would have been interesting to present the autocorrelation functions as a function of time.

Page 9, lines 298-310: Since DLS is much more sensitive to the presence of larger aggregates than smaller nanoparticles, it is difficult to conclude that larger structures are predominant. However, their presence is less significant in Figure 5(b) than in Figure 5(a).

Did you carry out any microscopy analysis of your samples? TEM, cryo-TEM or AFM images would have enable to confirm the size distribution of the self-assemblies. Besides, it would have been a plus in studying the self-assembly behavior of your systems.

Throughout the article, the authors claim that larger self-assemblies may correspond to micellar aggregates or vesicles, especially on page 7 line 270. I am not convinced by the formation of vesicles. Could you please develop this affirmation? Do you have other analyses to prove it, including microscopy analyses?

 

Author Response

We are grateful to the Reviewer for the comments, suggestions and criticisms. They were taken into account and we revised our manuscript accordingly. All the changes are given in red color.

 

Reviewer 1

This paper, by Plachouras et al., deals with the synthesis of amphiphilic diblock copolymers of poly(N-vinyl pyrrolidone) and poly(vinyl esters) bearing N-alkyl side chains by RAFT polymerization. Different copolymers with various poly(vinyl esters) (PVEs) blocks, molecular weights and compositions were synthesized. The self-assembly behavior of these copolymers in aqueous solution was then studied by DLS and SLS. Lastly, the encapsulation of hydrophobic curcumin and indomethacin within the core of the self-assemblies was studied in aqueous solution. Each step of the work is well detailed. The manuscript is well written and easy to read. However, some points need to be clarified. Therefore, I recommend its publication in Polymers after the following major revisions:

Even though the synthesis of the copolymers has already been published, it would have been good to remind the main synthesis steps in the SI and to show some data from the best systems in the SI, such as NMR spectra and GPC traces.

We added more details about the synthesis and the characterization of the samples in the SI according to the suggestion of the Reviewer.

Page 4 Table 1: “seize” should be replaced by “size” in the caption below Table 1.

We are sorry for the mistake. It was corrected in the text.

Page 4 line 136: Please precise how the water was added to the vial. Did you use a syringe pump? What was the flow rate?

No, we did not use a syringe pump to add the water in the vials. It is not a crucial procedure. The 20 ml of water were added gradually within a period of 5 min under constant stirring. The procedure gave equilibrium structures, since the experiments were repeated starting from different initial copolymer concentrations. The collected data showed similar results in all cases.

Page 6 Table 2: What is the difference between R_h0, R_h1,0, and R_h2,0?

The R_h1,0, and R_h2,0 values correspond to the hydrodynamic radii of each of the two populations, which exist in equilibrium, as were found by CONTIN analysis. The index “0” refers to the intercept at infinite dilution, taken from the plot of apparent R_h1 and R_h2 values vs concentration. The R_h0 values correspond to the results of the cumulant analysis, which is not able to differentiate the two populations. As previously mentioned, the index “0” refers to the R_h value at infinite dilution. This point was clarified in the text.  

Page 7 line 248-249: How do you estimate the fraction of micellar aggregates and therefore their increase? Same question for the percentage ranges from 65% to 80% mentioned in line 257 for samples PNVP-b-PVBu #3, #4 and #5?

CONTIN analysis is able to provide integration of the separate peaks corresponding to the different populations. Therefore, we have an estimation of the contribution of each population of particles in the solution.

Besides, could you please precise at which concentration the DLS results presented in Table 2, Table 3, Table 4 were obtained?

From the representative plots with the DLS data it is easy to check the range of concentrations that were employed. However, for the sake of clarity and as the Reviewer suggested we incorporated this information in the text.

Figure 1, Figure 3, Figure 4, and Figure 6 are not commented on.

In these Figures we provided typical examples from the DLS measurements. The basic conclusions were discussed in the text, as for all the samples. A few additional and more specific comments were added in the text.

For the DLS analyses, it would have been interesting to present the autocorrelation functions as a function of time.

The autocorrelation functions are the immediate result from the DLS measurements but it is not easy to interpret without using suitable methods of analysis (cumulants, CONTIN etc.). We added examples from the autocorrelation functions in the SI section.

Page 9, lines 298-310: Since DLS is much more sensitive to the presence of larger aggregates than smaller nanoparticles, it is difficult to conclude that larger structures are predominant. However, their presence is less significant in Figure 5(b) than in Figure 5(a).

The Reviewer is right. The large aggregates have a much more pronounced contribution to the scattering intensity. Therefore, CONTIN analysis overestimates their content in solution. However, the peak integration by CONTIN analysis shows a much higher contribution of the large aggregates. This result cannot be overcome considering the difference in size between the particles of the two populations. This result was further confirmed by the TEM measurements showing that an appreciable amount of the sample forms larger aggregates. 

Did you carry out any microscopy analysis of your samples? TEM, cryo-TEM or AFM images would have enable to confirm the size distribution of the self-assemblies. Besides, it would have been a plus in studying the self-assembly behavior of your systems.

TEM analysis was recently conducted and representative results were included in the manuscript, as the Reviewer suggested. The picture that was drawn by the DLS measurements was confirmed by TEM images. More details and comments were added in the text.

Throughout the article, the authors claim that larger self-assemblies may correspond to micellar aggregates or vesicles, especially on page 7 line 270. I am not convinced by the formation of vesicles. Could you please develop this affirmation? Do you have other analyses to prove it, including microscopy analyses?

The Reviewer is right. After performing the TEM analysis among the two possibilities of having vesicular structures or micellar aggregates we can safely exclude the first possibility. This comment was added in the text.

 

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript by Plachouras et al., investigates the self-assembly of amphiphilic PNVP-b-poly(vinyl ester) copolymers bearing N-alkyl side chains in water and their capacity to encapsulate hydrophobic drugs. It was reported that PVBu and PVDc form both small micelles and larger aggregates/vesicles, while PVSt forms only a single population of highly aggregated micelles. They also show that drug loading generally increases with increasing hydrophobic block content, but in PVSt, a plateau is observed due to restrictions imposed by the formation of crystalline domains of the micellar core. However, several issues should be addressed before publication:

1. The current Introduction does not provide enough background on how the nature of the hydrophobic block, the length of alkyl side chains, the copolymer composition, and the molecular weight influence the self-assembly behavior of amphiphilic block copolymers. This part should be strengthened.
2. The identification of micelles, aggregates, and vesicles relies solely on DLS data, which is far from convincing. Cryo-TEM/TEM, or SAXS/SANS measurements should be provided to confirm the type of assembly and morphology. The presence or absence of core-shell type structures can also be verified by small-angle scattering analysis.
3. The authors interpret negative kD values as evidence that interparticle hydrodynamic interactions prevail, and positive kD values as thermodynamically driven self-assembly. But I don’t think this is scientifically correct based on the definition of kD, since it does not reflect thermodynamic nature of the system. I would suggest the authors to reconsider and rephrase their interpretation.
4. Following my previous comments, instead of relying solely on DLS/SLS analysis, the interparticle interactions can be better quantified through SAXS or SANS measurements, where the scattering profiles can be analyzed with simple interaction models (e.g., hard-sphere or others). Such an approach can provide the structure factor independently of hydrodynamic contributions.
5. In the Conclusion (line 549), the authors claim that “Comparison among the three PNVP-b-PVEs copolymer families revealed that PNVP-b-PVBu samples displayed the highest loading capacity and efficiency.” However, this conclusion seems to be inconsistent with their results particularly for indomethacin.
6. In all the tables, D0 values has units of cm/s^2, which are incorrect.
7. In line 144, the authors claim that “The instrument was equipped with a Cyonics Ar+ laser operating at 640 nm”. However, this wavelength seems to be unrealistic for an Ar+ laser.
8. For Figures 1, 3, and 6, the current figure captions, i.e., “DLS plot…”, are too vague and does not explain what the data represent or why the linear fits are shown. The authors should also include error bars and discuss the fitting quality.
9. Since hydrophobic block length is expected to greatly affect CMC, the CMC values should be determined experimentally in this study.
10. In line 346, the authors state that “The long alkyl side chains of PVSt are able to form crystalline domains leading to the formation of compact micellar cores.” However, no experimental evidence or literature citation is provided to support this claim. Since this explanation is central to the interpretation of the drug loading plateau, direct evidence should be included.

Author Response

We thank the Reviewer for the valuable comments, suggestions and criticisms. The manuscript was revised accordingly and the changes in the text are given in red color.

Reviewer 2

The manuscript by Plachouras et al., investigates the self-assembly of amphiphilic PNVP-b-poly(vinyl ester) copolymers bearing N-alkyl side chains in water and their capacity to encapsulate hydrophobic drugs. It was reported that PVBu and PVDc form both small micelles and larger aggregates/vesicles, while PVSt forms only a single population of highly aggregated micelles. They also show that drug loading generally increases with increasing hydrophobic block content, but in PVSt, a plateau is observed due to restrictions imposed by the formation of crystalline domains of the micellar core. However, several issues should be addressed before publication:

1. The current Introduction does not provide enough background on how the nature of the hydrophobic block, the length of alkyl side chains, the copolymer composition, and the molecular weight influence the self-assembly behavior of amphiphilic block copolymers. This part should be strengthened.

The requested information by the Reviewer was added in the Introduction of the manuscript.

1. The identification of micelles, aggregates, and vesicles relies solely on DLS data, which is far from convincing. Cryo-TEM/TEM, or SAXS/SANS measurements should be provided to confirm the type of assembly and morphology. The presence or absence of core-shell type structures can also be verified by small-angle scattering analysis.

The microscopy facility was very recently established in our department. Therefore, we performed TEM measurements and representative results are included in the revised version of our manuscript. These images conform that we have in most cases equilibrium between micelles and micellar aggregates. Unfortunately, it is not easy to conduct SAXS or even worse SANS measurements.

1. The authors interpret negative kD values as evidence that interparticle hydrodynamic interactions prevail, and positive kD values as thermodynamically driven self-assembly. But I don’t think this is scientifically correct based on the definition of kD, since it does not reflect thermodynamic nature of the system. I would suggest the authors to reconsider and rephrase their interpretation.

It is known that  reflects both thermodynamic and hydrodynamic interactions, since it is related to the second virial coefficient  and the friction coefficient according to the equation:  where  is the molecular weight,  the concentration dependence of the friction coefficient, and  the polymer’s partial specific volume. Positive k_D values indicate high aggregation numbers or strong thermodynamic interactions, whereas low or even negative k_D values is a manifestation of low aggregation numbers and strong hydrodynamic interactions.

 

1. Following my previous comments, instead of relying solely on DLS/SLS analysis, the interparticle interactions can be better quantified through SAXS or SANS measurements, where the scattering profiles can be analyzed with simple interaction models (e.g., hard-sphere or others). Such an approach can provide the structure factor independently of hydrodynamic contributions.

Unfortunately, we do not have in our department a SAXS facility and of course it is even more difficult to perform SANS experiments, since neutron source is required.

1. In the Conclusion (line 549), the authors claim that “Comparison among the three PNVP-b-PVEs copolymer families revealed that PNVP-b-PVBu samples displayed the highest loading capacity and efficiency.” However, this conclusion seems to be inconsistent with their results particularly for indomethacin.

The main conclusions are that: a) all samples have a significantly high drug loading capacity and efficiency and b) indomethacin has a much higher ability of encapsulation than curcumin. We are running these days initial experiments with release studies of drugs from these and other similar micellar systems based on PNVP materials. The general conclusions are that with PVEs with short alkyl side chains the DLC and DLE values either increase or at least remain stable upon increasing the amount of the drug, whereas with PVEs with long alkyl side chains DLC and DLE decrease in the same sense. This is due to the stronger interaction of the long side chains and the ability to form crystalline domains that restrict the incorporation of higher amounts of drug. From our initial release studies, it seems that the kinetics of release is more pronounced for copolymers with short PVEs chains. 

1. In all the tables, D0 values has units of cm/s^2, which are incorrect.

We are sorry for the mistake. The correct units (cm²/s) were given in the revised text.

1. In line 144, the authors claim that “The instrument was equipped with a Cyonics Ar+ laser operating at 640 nm”. However, this wavelength seems to be unrealistic for an Ar+ laser.

We are sorry for the mistake. The Cyonics Ar+ laser operates at 488 nm and refers to another instrument of our lab. The correct data were added in the text.

1. For Figures 1, 3, and 6, the current figure captions, i.e., “DLS plot…”, are too vague and does not explain what the data represent or why the linear fits are shown. The authors should also include error bars and discuss the fitting quality.

The captions were revised and the error bars were introduced in the plots. Comments on the linearity of the plots were included in the text.

1. Since hydrophobic block length is expected to greatly affect CMC, the CMC values should be determined experimentally in this study.

The CMC values consist an important parameter in micellization studies. However, we did not proceed with the determination of the CMC values for the following reasons: a) for the encapsulation studies we have to work well above the CMC value, which was verified in this study. Therefore, the knowledge of the exact CMC values is not crucial for this work. b) The same samples were employed for micellization studies in tetrahydrofuran, which is a selective solvent for the PVEs blocks. For each manuscript we had to perform additional experiments, asked by the Reviewers and consequently we do not have any more quantities of the samples for more experiments.

1. In line 346, the authors state that “The long alkyl side chains of PVSt are able to form crystalline domains leading to the formation of compact micellar cores.” However, no experimental evidence or literature citation is provided to support this claim. Since this explanation is central to the interpretation of the drug loading plateau, direct evidence should be included.

It is known that polymers bearing long alkyl side chains (with more than 12 carbon atoms) are able to form crystalline domains. In any Polymer Handbook we can find the Tm values of these polymers [e.g. poly(stearyl methacrylate) and poly(stearyl acrylate), polystyrenes substituted at the p-position with long alkyl groups etc.]. We included a relevant paper on the micellization behavior of block copolymers of polystyrene and poly(stearyl methacrylate).

 

 

Reviewer 3 Report

Comments and Suggestions for Authors

The problem of poor bioavailability of water-insoluble drugs is a significant challenge, making the article "Amphiphilic Diblock Copolymers of Poly(N-Vinyl Pyrrolidone) and Poly(Vinyl Esters) Bearing N-Alkyl Side Chains for the En-capsulation of Curcumin and Indomethacin " by Nikolaos V. Plachouras et al. highly relevant. The work is conducted at a high experimental level, employing a range of research methods.

 

However, I have several comments and suggestions regarding this study:

1. Figures 1, 3, 4, 6, and 8 lack error bars. Could the authors clarify if the experiments, the results of which are presented in these figures, were performed only once?
2. Tables 7 and 8 are quite large and complex, making them difficult to interpret. It might be beneficial to present the key comparative data from these tables in the form of bar charts for better clarity and visual impact.
3. Unfortunately, the authors did not clearly state the specific aims and objectives of the work in the introduction. I would recommend explicitly outlining the research goals.
4. In the "Conclusions" section, the authors state that "the amphiphilic block copolymers investigated in this study demonstrate significant potential as carriers for drug delivery applications." However, this claim appears overly optimistic without supporting data on drug release profiles from the block copolymers. I recommend either moderating this conclusion or acknowledging the need for future release studies.

Comments on the Quality of English Language

To maximize the text's compliance with English-language scientific publication standards, please consider having it undergo final proofreading and editing by a native speaker or an editor with exceptional English proficiency (C1/C2 level). This will enhance the natural flow of the language and correct any minor imperfections.

Author Response

We thank the Reviewer for the fruitful comments. We took into consideration these comments and we revised our text. All the changes are given in red color.

Reviewer 3

The problem of poor bioavailability of water-insoluble drugs is a significant challenge, making the article "Amphiphilic Diblock Copolymers of Poly(N-Vinyl Pyrrolidone) and Poly(Vinyl Esters) Bearing N-Alkyl Side Chains for the Encapsulation of Curcumin and Indomethacin " by Nikolaos V. Plachouras et al. highly relevant. The work is conducted at a high experimental level, employing a range of research methods.

 

However, I have several comments and suggestions regarding this study:

1. Figures 1, 3, 4, 6, and 8 lack error bars. Could the authors clarify if the experiments, the results of which are presented in these figures, were performed only once?

Each measurement was conducted ten (10) times and the presented results are the average values. As the Reviewer requested the error bars were added in the Figures and the revised Figures are now included in the manuscript.

1. Tables 7 and 8 are quite large and complex, making them difficult to interpret. It might be beneficial to present the key comparative data from these tables in the form of bar charts for better clarity and visual impact.

Tables 7 and 8 along with 9 and 10 provide the cumulative results from the encapsulation experiments of curcumin and indomethacin respectively. Similar data are given in almost every paper referring to encapsulation experiments. The results for each sample are fully separated and it is not difficult to follow. Splitting the data to more Tables or giving bar charts would result in too many figures and in a very extended manuscript (consider also the new results from the TEM measurements that are included in the text).

1. Unfortunately, the authors did not clearly state the specific aims and objectives of the work in the introduction. I would recommend explicitly outlining the research goals.

The research goals of this work were actually outlined at the end of the Introduction section. However, more details were added as the Reviewer suggested.

1. In the "Conclusions" section, the authors state that "the amphiphilic block copolymers investigated in this study demonstrate significant potential as carriers for drug delivery applications." However, this claim appears overly optimistic without supporting data on drug release profiles from the block copolymers. I recommend either moderating this conclusion or acknowledging the need for future release studies.

We employed the words “significant potential”. This comes from the satisfactory encapsulation ability of our systems. However, the delivery process is something different. We are in progress to perform delivery studies from these and other systems that have been employed for drug encapsulation. The initial results from delivery studies based on the copolymer PNVP-b-PVSt #3 are given below. A dialysis bag was employed for the release of indomethacin in a buffer solution of pH=5.0. The release plot is given below, showing that after 24 hours almost 40% of indomethacin has been released from the micellar system. It seems that the PVSt based systems, due to their ability to form crystalline cores do not show highly fast release profiles. 

 

Sample	Ind. 1 (w/w)	A 2 (a.u.)	Pol. 3 (μg/mL)	DIF 4 (μg/mL)	LD 5 (μg/mL)	DLC (%)	DLE (%)
PNVP-b-PVSt #3	10%	0.50	1,931.65	195.27	220.45	11.43	113.03

 

Time (h)	% Drug Release
1	6.72
3	13.90
6	26.39
12	34.24
24	36.68
48	37.15

 

 

Comments on the Quality of English Language

To maximize the text's compliance with English-language scientific publication standards, please consider having it undergo final proofreading and editing by a native speaker or an editor with exceptional English proficiency (C1/C2 level). This will enhance the natural flow of the language and correct any minor imperfections.

The manuscript was checked and corrected by a native in the USA scientist.

 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have clearly and satisfactorily answered to the raised questions. I recommend this revised version for publication in Polymers.

Reviewer 2 Report

Comments and Suggestions for Authors

I appreciate the efforts from the authors to revise the manuscript and address my previous comments. While some of my concerns, particularly those related to structural characterization and data interpretation, remain only partially resolved, I recognize that the authors have made some reasonable attempts and responses. The overall quality of the revised manuscript is not perfect but somewhat acceptable, and I defer to the judgments by the academic editor or other reviewers regarding whether the manuscript in its current form meets the publication standards of the journal.

Reviewer 3 Report

Comments and Suggestions for Authors

The authors took the comments into account and revised the article. I recommend accepting it for publication.