Water-in-Water Emulsion as a New Approach to Produce Mesalamine-Loaded Xylan-Based Microparticles
, Francisco Alexandrino, Jr. 3, Silvana C. C. Urtiga 1, Karen C. H. Silva 4, Daniel C. F. Soares 5 and Eryvaldo S. T. Egito 1,3,*Round 1
Reviewer 1 Report
In this manuscript, the authors developed a water-in-water emulsion approach to produce mesalamine-loaded xylan-based microparticles. The microparticles were prepared without introducing harmful solvents and the model drug was loaded at drug loading efficiency as high as 49.6% and released in a controlled manner. The publication of this work could benefit some researchers in this field. However, some changes need to be made to improve the readability and quality of the manuscript. The comments and questions are listed below.
1. The authors had better provide a schematic figure for the water-in-water emulsion process for the microparticle fabrication.
2. In Figure 1, what do the “X1”, “X2”, “Y1” and “Y2” stand for?
3. In Figure 2B, huge microparticles with diameter around 50 um could be observed. And the two big microparticles in Figure 2B might have a weight 5 time higher than all the small microparticles combined. But the size of the microparticles is given as 4.8 um due to the size frequency in Figure 2D. My question is whether the particle number or the particle weight contribute more to the drug loading and drug release behavior? Is it reasonable to regard the drug loading and release behavior belong to the 4.8 um microparticles.
4. In Figure 3, CaCl2 should be changed to “CaCl2”.
5. There are some grammar errors in the manuscript. In the abstract, line 23, “microparticles… was…”.
Author Response
In this manuscript, the authors developed a water-in-water emulsion approach to produce mesalamine-loaded xylan-based microparticles. The microparticles were prepared without introducing harmful solvents and the model drug was loaded at drug loading efficiency as high as 49.6% and released in a controlled manner. The publication of this work could benefit some researchers in this field. However, some changes need to be made to improve the readability and quality of the manuscript. The comments and questions are listed below.
We are glad you have liked our manuscript. Thank you for the important remarks that made it much better.
1. The authors had better provide a schematic figure for the water-in-water emulsion process for the microparticle fabrication.
As per your request, a flow-chart about the production of XBM and XBM-Ca was added. We believe that now the process is more comprehensible for the readers.
Figure 1. Flow chart of the production of XBM and XBM-Ca.
2. In Figure 1, what do the “X1”, “X2”, “Y1” and “Y2” stand for?
The authors are thankful to you for bringing up this point. A description of the required parameters was added in Figure 1 (now Figure 2 on this R1-Version) legend on lines 194 - 197.
Text Added text
“…X1 stands for the xylan dispersion at 40%(w/v), X2 is the PEG enriched solution 32%(w/v), Y1 and Y2 represent the volume ratio of the two phases for the formation of the emulsions.”
3. In Figure 2B, huge microparticles with diameter around 50 um could be observed. And the two big microparticles in Figure 2B might have a weight 5 time higher than all the small microparticles combined. But the size of the microparticles is given as 4.8 um due to the size frequency in Figure 2D. My question is whether the particle number or the particle weight contribute more to the drug loading and drug release behavior? Is it reasonable to regard the drug loading and release behavior belong to the 4.8 um microparticles.
The authors agree with you that big particles can be seen in the optical microscopy. Also, we are aware of the high polydispersion on the XBM and XBM-Ca formulations, which is shown on the particle size distribution (now Fig. 3C and 3D on this R1 Version). It is possible that the presence of big particle might have an impact over the drug loading and its release from the microparticles. At this point in the research, we focused on the development of the microparticles and their ability to entrap the 5-ASA. On the future, we will take into account your remark and we will try to separate the microparticles fraction for evaluation of their release profile. However, it is important to note that the release profile was not quite disturbed for such microparticles.
4. In Figure 3, CaCl2 should be changed to “CaCl2”.
Corrected, thank you. It is important to note that now this figure is numbered as 4.
Figure 4: Scheme of the crosslinking reaction between xylan (Xyl), CaCl2 and STMP. Reaction (1) represents only the chemical bond between STMP-Xyl. Reaction (2) represents the process of chemical crosslinking between Ca2+-Xyl-STMP, respectively, and Reaction (3) represents the ionic interaction between Ca2+-Xyl.
5. There are some grammar errors in the manuscript. In the abstract, line 23, “microparticles… was…”.
The authors are grateful to you for bringing up this point. An entire revision of the English was performed on this R1-Version of the manuscript.
Reviewer 2 Report
In this manuscript, the authors present xylan microparticles loaded with mesalamine via the water-in-water emulsification. The morphology of particles was characterized with optical microscope and the encapsulation was studied using FTIR and XRD. Finally, release of drug from the was evaluated in vitro. Publication in this journal is recommended after the following concerns are address:
1. In line 226~230, the authors hypothesized that CaCl2 perform a role as a crosslinker, thus the compactness of the produced microparticles increases. However, XBM-Ca-F7 is bigger than XBM-Ca-F1 regardless of the increased amount of CaCl2.
2. In figure 2, it is hard to tell the difference in both images. Please provide improved quality of images.
3. In line 281, The authors mentioned that the shielding effect from PEG was not shown in formulations containing CaCl2. Please give more detailed explanation.
4. In figure 5, please add mark on the characteristic peaks on IR spectra such as P=O bond, etc.
5. What are X1,X2,Y1,and Y2 in Figure 1? Please give footnote.
6. Can XBM particles be dispersed in water without shape change?
7. Typo: in line 305, “XBM-Ca-F9 loaded with 5-ASA.”->“XBM-Ca-F9 loaded with 5-ASA (I).”
Author Response
Reviewer 2
In this manuscript, the authors present xylan microparticles loaded with mesalamine via the water-in-water emulsification. The morphology of particles was characterized with optical microscope and the encapsulation was studied using FTIR and XRD. Finally, release of drug from the was evaluated in vitro. Publication in this journal is recommended after the following concerns are address:
We are glad you have liked our manuscript and pointed out that it deserves to be published at the Applied Sciences Journal. Thank you for the important remarks that made it still better.
1. In line 226~230, the authors hypothesized that CaCl2 perform a role as a crosslinker, thus the compactness of the produced microparticles increases. However, XBM-Ca-F7 is bigger than XBM-Ca-F1 regardless of the increased amount of CaCl2.
The authors are thankful for this remark and agree with you that the XBM-Ca-F7 has a bigger average particle size than XBM-Ca-F1. However, our statement made between lines 226 – 230 tried to make a comparison between the formulations in which CaCl2 was added (XBM-Ca-F1 to XBM-Ca-F9) and the ones without CaCl2 (XBM). On the other hand, looking to our data on Table 1, the parameters chosen to evaluate the production process could not predict the observed behavior between XBM-Ca-F7 and XBM-Ca-F9 (although the Response surface plot (Figure 5) indicated some correlation). Moreover, the general idea that we would like to point out is that microparticles without CaCl2 has bigger size. I hope you agree with such statement. Moreover, we clarify such approach by adding some text on lines 240-241 of the R1-Version of the manuscript.
2. In figure 2, it is hard to tell the difference in both images. Please provide improved quality of images.
We are sorry that you didn’t like the images of Figure 2 (now Figure 3 on this R1 – Version of the manuscrip). In fact, to me the images sound clear and we think that all the comments made on the manuscript body relay in what can be seem on it. However, we work on the microscopy image to improve its sharpness and added it to this R1-Version of the manuscript.
3. In line 281, The authors mentioned that the shielding effect from PEG was not shown in formulations containing CaCl2. Please give more detailed explanation.
That is an interesting point. We have no clue to answer you this question, based on evidences. However, we believe that CaCl2 can be able to induce some changes at the interface of the microparticles. Indeed, it could be speculated that the CaCl2 play an important role at the interface of the microparticles, hindering the shielding effect of PEG. We added this statement on lines 294 and 295 of the R1-Version of the manuscript.
4. In figure 5, please add mark on the characteristic peaks on IR spectra such as P=O bond, etc.
We are sorry to have induced you for such mistake. In fact, the P=O bond was not seeming in all formulations, as we stated from line 296 on this R1 Version of the manuscript. However, because we didn’t start a new paragraph, it seemed that we were talking about the reasons of the hindering shielding effect for CaCl2 microparticles. We correct the text to make this clear.
5. What are X1, X2, Y1, and Y2 in Figure 1? Please give footnote.
The authors are thankful to you for bringing up this point, as also made the Reviewer 1. A description of the required parameters was added in Figure 1 legend (now Figure 2 on this R1-Version of the manuscript) on lines 197 - 199.
Text Added text
“…X1 stands for the xylan dispersion at 40%(w/v), X2 is the PEG enriched solution 32%(w/v), Y1 and Y2 represent the volume ratio of the two phases for the formation of the emulsions.”
6. Can XBM particles be dispersed in water without shape change?
The XBM may be re-dispersed in small amounts of aqueous media, as it was performed for the morphological characterization. However, this formulation was designed to be used on its dried form by its compression with inactive ingredients or through its encapsulation into hard gelatin capsules. The morphological evaluation into high diluted media, such as in the in vitro drug release, has not been performed. by our group.
7. Typo: in line 305, “XBM-Ca-F9 loaded with 5-ASA.” à “XBM-Ca-F9 loaded with 5-ASA (I).”
Thank you to highlight this mistake. A review on the English grammar, including typos, was performed to this R1-version of the manuscript.
Reviewer 3 Report
The reviewed work describes a new technique of encapsulation of 5-aminosalicylic acid (5-ASA), a drug used in diseases of the intestine, mainly the colon; inflammatory bowel disease, including ulcerative colitis and Crohn's disease. The choice of kind of material from which microcapsules were formed - a polysaccharide undergoing degradation under the influence of enzymes in the large intestine is therefore very deliberate. The work is interesting, mainly due to the results obtained. The investigations and their description are carried out at a very good level. In my opinion, the work should be published. However, the manuscript should be a bit complemented, the authors should introduce additional explanations and descriptions;
- Describe the obtained capsule stability, the ability and efficiency of separation from the aqueous phase, the possibilities and conditions of transport and storage.
- In part 3.4. Chemical characterization of XBM and XBM-Ca by ATR-FTIR has no practically conclusions regarding the interaction between the matrix and the drug (check the existence of hydrogen bonds). Such conclusion based on a more detailed analysis of the obtained FTIR results should be placed in this part of the manuscript
- Similarly in part 3.5. XRD analysis is missing the final summary conclusion of the discussion on the obtained results from studies on changes in crystallinity
- The authors argue that " is the first time that a low molecular weight drug has been successfully encapsulated into microparticles produced by this technique, which broads the application of this technology". It will be so if the presented method of encapsulation apply to other drugs used in colorectal therapies (eg such as: tinidazol, metronidazole, infliksymab) . If so, which drugs can be use probably ? - Please enter this short discussion in the part - Conclusions.
Author Response
The reviewed work describes a new technique of encapsulation of 5-aminosalicylic acid (5-ASA), a drug used in diseases of the intestine, mainly the colon; inflammatory bowel disease, including ulcerative colitis and Crohn's disease. The choice of kind of material from which microcapsules were formed - a polysaccharide undergoing degradation under the influence of enzymes in the large intestine is therefore very deliberate. The work is interesting, mainly due to the results obtained. The investigations and their description are carried out at a very good level. In my opinion, the work should be published. However, the manuscript should be a bit complemented, the authors should introduce additional explanations and descriptions;
We are glad you have liked our manuscript and pointed out that it deserves to be published at the Applied Sciences Journal. As you can see, we added the additional explanations requested by you, and we hope that now the manuscript is much better.
1. Describe the obtained capsule stability, the ability and efficiency of separation from the aqueous phase, the possibilities and conditions of transport and storage.
Thank you for bringing the discussion about the stability of the microparticles. Indeed, this is an important point, once we wish the pharmaceutical usage of these microparticles. However, at this point of our research we do not have well-established (meaning, at the conditions preconized by the ICH) stability studies, as well as the characterization of the drying process for the XBM-Ca-F9 microparticles (meaning, the absorption/desorption process of water from the powder). Nonetheless, when it comes to the separation of the aqueous phase during the production of the XBM-Ca-F9 formulation, it was considered efficient, by naked eye, once the liquid used for the cleaning steps did not reveal changes in the viscosity, which would mean the presence of PEG chains in the medium. Furthermore, from storage and transportation point of view, it is not expected that this powder will have any disadvantage when compared to other polysaccharide powder used in the pharmaceutical industry (e.g.: xanthan gum and starch).
2. In part 3.4. Chemical characterization of XBM and XBM-Ca by ATR-FTIR has no practically conclusions regarding the interaction between the matrix and the drug (check the existence of hydrogen bonds). Such conclusion based on a more detailed analysis of the obtained FTIR results should be placed in this part of the manuscript
The authors agree with you about that. However, as stated in the text, we were not able to see many chemical bonds in the XBM-Ca-F9 spectra, thus, leading us to the hypothesis of the PEG shielding effect. Additionally, the technique applied for the production was not designed to provide any linkage between the polymeric chains and the drug itself. Also, the results observed in the in vitro drug release study leads to an interpretation that no strong bond (such as covalent linkages) was formed during the production. We improved the discussion about some peak changes, occurred in the XBM-Ca-F9, that may be related to the enhancement of the 5-ASA loading. The changes made are described in lines 303-305 of the R1-Version of this manuscript.
Text added:
“In addition, the increase on the intensity of the peak near 1500 cm-1 may be associated with the enhancing of drug 5-ASA loading, once these peaks are associated with C-C and C=C stretching mode, and also N-H bond [22].”
3. Similarly in part 3.5. XRD analysis is missing the final summary conclusion of the discussion on the obtained results from studies on changes in crystallinity.
We tried to discuss a little bit more the data from the XRD analysis. Unfortunately, a deep discussion could not be performed because we have no access to high performance equipment such as NMR. Therefore, the finding of the presence of similar peaks in the XBM-Ca-F9 formulation when compared to the mesalamine crystalline structure was surprising. On the other hand, the mesalamine was later identified in the in vitro drug release assay, implying in a non-covalently bond interaction. An additional sentence was added to the paragraph in other to better correlate the XRD data with other results of the manuscript. Please, check lines 319 and 320.
Text added:
“The presence of the peaks from 5-ASA on the XBM-Ca-F9 diffractogram reveals the association of the 5-ASA with the xylan through physical interactions, once no new bound was identified in the ATR-IR analysis, only an enhancement of the peaks at the region between 1400 – 1600 cm-1, as previously discussed.”
4. The authors argue that " is the first time that a low molecular weight drug has been successfully encapsulated into microparticles produced by this technique, which broads the application of this technology". It will be so if the presented method of encapsulation apply to other drugs used in colorectal therapies (eg such as: tinidazol, metronidazole, infliksymab). If so, which drugs can be use probably? - Please enter this short discussion in the part - Conclusions.
Thank you to highlight other possibilities of the use of our system. In fact, we would have described other drugs that would benefit from this technology. However, the addition of a deep discussion about that would not be based on the results, but rather speculative. Noteworthy, we tried to improve the conclusion by adding a last sentence on the Conclusion Section.
Reviewer 4 Report
Souza et al. have submitted a well-written manuscript that rises the important chalenge of encapsulation of small drugs into emulsion-templated microparticles. Significantly, the authors probed water-in-water emulsions that are free of harmful organic solvents. These systems are highly advantageous and are widely used, yet the understanding of physico-chemical phenomena occuring in these emulsions is still poor.
The study is well-organised, preparedemulsions are well-charachterised, all the conclusions are supported by experimental findings.
I recommend the manuscript for the publication after minor corrections:
Section Materials: specify the quality/source of water used in the study.
Lines 114-115: "... a previously validated method..." provide a reference.
Line 307: correct to "Table 1".
Paragraph started from line 323: mathematical modelling performed i nthis study is poorly described. Add and discuss other models (besides of the best fitted Peppas model) that have been used. Supplement the manuscript with corresponding equations, fitting parameters and R2-values.
Author Response
Reviewer 4
Souza et al. have submitted a well-written manuscript that rises the important chalenge of encapsulation of small drugs into emulsion-templated microparticles. Significantly, the authors probed water-in-water emulsions that are free of harmful organic solvents. These systems are highly advantageous and are widely used, yet the understanding of physico-chemical phenomena occuring in these emulsions is still poor.
The study is well-organised, prepared emulsions are well-charachterised, all the conclusions are supported by experimental findings.
I recommend the manuscript for the publication after minor corrections:
We are glad you liked our manuscript and pointed out the minor corrections to make it still better.
1- Section Materials: specify the quality/source of water used in the study.
As requested, we added the details of the water source on the new R1-Version of the manuscript. Please, check the lines 77 – 79.
Text added:
“Water (conductivity ≤ 3.5 ?S), obtained from deionization followed by reverse osmosis process, was used to produce the microparticles and prepare the dissolution medium. All chemicals were of analytical grade and used as received.”
2 - Lines 114-115: "... a previously validated method..." provide a reference.
Although validated, the used method was not yet published. Therefore, we made some changes on the text to avoid misunderstandings. Please, check lines 119 – 120 of the R1-version of the manuscript.
3 - Line 307: correct to "Table 1".
Paragraph started from line 323: mathematical modelling performed in this study is poorly described. Add and discuss other models (besides of the best fitted Peppas model) that have been used. Supplement the manuscript with corresponding equations, fitting parameters and R2-values.
As requested, we deeply discussed the choice of the model and created a Supplementary Material file to address this comment. Please, check lines 342 – 351 of the R1-version of the manuscript.
Text added:
“In order to understand the mechanism on the initial release of 5-ASA from XBM-Ca-F9, some mathematical models were applied to the data. Initially, a first attempt was made to fit the data to a concentration independent model (e.g.: linear model). However, the drug was not released in a constant rate. Thus, models that considered a non-linear release were tested. The Higuchi, Korsmeyer-Peppas and Peppas-Shalin models were, then, tried. The Higuchi model showed a poor adjustment to the data, probably due to the non Fickian diffusion of the drug through the polymeric matrix, as it will be later discussed. On the other hand, the Korsmeyer-Peppas and the Peppas-Shalin model, which consider the Fickian diffusion and the relaxation of the polymeric chains, were able to best fit the data. Therefore, the Peppas-Sahlin model with t-lag was the one with best fitting parameters, once it considers a lag time for the beginning of the drug release (See Table S1) [29,32].”
Round 2
Reviewer 2 Report
The authors have revised their manuscript accordingly. Thereby, I recommend its publication in this journal .
