Drug-Containing Layered Double Hydroxide/Alginate Dispersions for Tissue Engineering

Round 1

Reviewer 1 Report

The research deals with the intercalation of naproxen and ibuprofen drug molecules into the interlamellar space of Mg₃Al-LDH particles dispersed in alginate solutions. The authors applied several characterization techniques (XRD, SEM-EDS, DLS, FT-IR and ELS), some part of the work is maybe of interest, especially the cytoprotective effects found. The topic is interesting and the readers will probably find the results useful, the authors showed abundant interesting results and gave reasonable interpretations, but several statements need to be addressed, the manuscript in its present form requires major revision.

11.     It is not clear why the sterilization by thermal treatment resulted in a significant increase in hydrodynamic diameters compared to the sonication, while the zeta potential values showed large fluctuations depended on the chemical quality of the intercalated drug molecules. It should be discussed.

22.     For the better visualization of the XRD and FT-IR measurements, the patterns of the pristine (chloride intercalated) Mg₃Al-LDH should be added to the Fig. 1b and 1a.

33.     In the introduction section, the alganite-LDHs composites are widely discussed because its fundamental importance, but, unfortunately, plenty of recent references are missing related to the intercalation/liberation of ibuprofen and naproxen molecules in LDH systems.

Synthesis and application of magnetic@layered double hydroxide as an anti-inflammatory drugs nanocarrier, J. Nanobiotechnol., 18 (2020) 155

Naproxen/layered double hydroxide composites for tissue-engineering applications: Physicochemical characterization and biological evaluation, Clays Clay Miner., 68 (2020) 623–631

Synthesis of layered double hydroxides intercalated with drugs for controlled release: Successful intercalation of ibuprofen and failed intercalation of paracetamol, J. Pharm. Sci., 110 (2021) 1779–1787

Conventional or mechanochemically-aided intercalation of diclofenac and naproxen anions into the interlamellar space of CaFe-layered double hydroxides and their application as dermal drug delivery systems, Appl. Clay Sci., 212 (2021) 106233

Fe(III)-based layered double hydroxides carrying model naproxenate anions: Compositional and structural aspects, ChemistrySelect, 7 (2022) e202103880

44.     The style of the Latin words used is not consistent, italic and non-italic modes alternate!

55.     The information content of the SEM imagines is small, the magnification of the relevant part of the pictures or using photos captured with higher magnifications would be advisable. 

66.     The in vitro release profiles well demonstrated the drug retention capacity of LDHs, but how can the generally lower release of the interlayer anions be interpreted despite the higher LDH dispersion concentration used? How the LDH concentration might be related to the ion exchange processes in the phosphate buffer solution?

Author Response

The research deals with the intercalation of naproxen and ibuprofen drug molecules into the interlamellar space of Mg₃Al-LDH particles dispersed in alginate solutions. The authors applied several characterization techniques (XRD, SEM-EDS, DLS, FT-IR and ELS), some part of the work is maybe of interest, especially the cytoprotective effects found. The topic is interesting and the readers will probably find the results useful, the authors showed abundant interesting results and gave reasonable interpretations, but several statements need to be addressed, the manuscript in its present form requires major revision.

 Thank your kind comments, they aided in improving the manuscript

11. It is not clear why the sterilization by thermal treatment resulted in a significant increase in hydrodynamic diameters compared to the sonication, while the zeta potential values showed large fluctuations depended on the chemical quality of the intercalated drug molecules. It should be discussed.

Effectively, the incorporation of LDH particles in Alg dispersions produced great changes in the zeta potential of the particles but not in their hydrodynamic diameter values. This behavior has been previously reported for polyelectrolytes that present low affinity for the LDH surface (and particularly Alg has been previously attached on the surface of LDH-Cl particles, see Ref. [19] ). In the case of drug intercalated LDH, due to their heavy aggregation and hydrophobic interactions between their particles, the effect was greater, showing significant diminutions in the zeta potential values while maintaining their aggregation. We have introduced new text in l. 337-345 to improve the discussion.

22. For the better visualization of the XRD and FT-IR measurements, the patterns of the pristine (chloride intercalated) Mg₃Al-LDH should be added to the Fig. 1b and 1a.

The figure has been corrected accordingly. New text has been added to introduce the new data in lines 230-232 and 260.

33. In the introduction section, the alganite-LDHs composites are widely discussed because its fundamental importance, but, unfortunately, plenty of recent references are missing related to the intercalation/liberation of ibuprofen and naproxen molecules in LDH systems.

Synthesis and application of magnetic@layered double hydroxide as an anti-inflammatory drugs nanocarrier, J. Nanobiotechnol., 18 (2020) 155

Naproxen/layered double hydroxide composites for tissue-engineering applications: Physicochemical characterization and biological evaluation, Clays Clay Miner., 68 (2020) 623–631

Synthesis of layered double hydroxides intercalated with drugs for controlled release: Successful intercalation of ibuprofen and failed intercalation of paracetamol, J. Pharm. Sci., 110 (2021) 1779–1787

Conventional or mechanochemically-aided intercalation of diclofenac and naproxen anions into the interlamellar space of CaFe-layered double hydroxides and their application as dermal drug delivery systems, Appl. Clay Sci., 212 (2021) 106233

Fe(III)-based layered double hydroxides carrying model naproxenate anions: Compositional and structural aspects, ChemistrySelect, 7 (2022) e202103880

The most relevant articles have been included in the reference list. Thanks for these enriching articles. The whole bibliography has been also revised.

44. The style of the Latin words used is not consistent, italic and non-italic modes alternate!

The use of italic mode has been revised to increase its uniformity.

55. The information content of the SEM imagines is small, the magnification of the relevant part of the pictures or using photos captured with higher magnifications would be advisable. 

We have included images with higher magnifications in figure 3.

66.    The in vitrorelease profiles well demonstrated the drug retention capacity of LDHs, but how can the generally lower release of the interlayer anions be interpreted despite the higher LDH dispersion concentration used? How the LDH concentration might be related to the ion exchange processes in the phosphate buffer solution?

From the results obtained it can be deduced that the rate determining step of drug release was the anion diffusion through the gel, which was not affected by LDH concentration in the case of PBS. It was diminished, in relative terms, in the case of SBF. Nevertheless, it is important to note that drug release profiles were expressed relatively, as a cumulative percentage of drug released at each time point respect to the total amount of drug in the tested sample. So, in absolute terms, the samples containing a larger concentration of LDH were already releasing a larger amount of drug anions.

Reviewer 2 Report

In this manuscript, the author proved that drug-LDH/Alg could control the release of drugs in simulated body fluid and improve the biocompatibility of the drugs and mitigate the cytotoxic effects of the pure drugs. Generally, the manuscript is organised and presented with a fine manner. However, the characteristic of drug-LDH/Alg was not clearly studied and illustrated. The following points need to be addressed to strengthen the manuscript:

1. line 68-69, "LDH could increase the drug loading capacity". How LDH improved drug loading capacity in the manuscript? Is the drug loading capacity presented in the manuscript?

2. In Fig 1, FTIR and XRD of LDH  should be added as the control. Peaks of LDH/Ibu/Nap should be marked.

3. The section of dispersion by sonication/thermal treatment does not seem to fit in the logic of the manuscript. I don't think they are worthy in the main text. It may be fine to put in SI though. And, what is PA? Note that all abbreviations should be clearly described at the first appearance.

4. DLS data are not reliable because the accuracy range of Delsa Nano C instrument can only reach 7 um, whereas your data were 10 um. This should be re-done with other proper facility.

5. SEM images are not acceptable at such scale bar. It is hard to tell if what is shown in LDH-Nap is the sample or dust. The LDH-Ibu image does not provide any valid information. They should be re-taken with better sample preparation and higher magnification with image of pure LDH as the control.

6. The formulation used for tissue engineering is drug-loaded LDH/alg, then the drug-loaded LDH/alg should be characterised with all above techniques as well, which are missing at the moment.

7. TEM is a "compulsory" tool when you study LDH and should be added.

8. Sustained drug release and prolonged drug retention time are essential for tissue regeneration, the author should provide data of drug release efficiency with longer time, like 48-72 hours.

9. How long can the drugs reside in the tissue for regeneration?

Author Response

In this manuscript, the author proved that drug-LDH/Alg could control the release of drugs in simulated body fluid and improve the biocompatibility of the drugs and mitigate the cytotoxic effects of the pure drugs. Generally, the manuscript is organised and presented with a fine manner. However, the characteristic of drug-LDH/Alg was not clearly studied and illustrated. The following points need to be addressed to strengthen the manuscript:

 Thank your kind comments, they aided in improving the manuscript

1. line 68-69, "LDH could increase the drug loading capacity". How LDH improved drug loading capacity in the manuscript? Is the drug loading capacity presented in the manuscript?

In lines 68-69 it is stated “…, they present high drug loading capacity of acidic drugs between their layers”; the phrase included by the reviewer was not found by us. The LDH indeed have a high drug loading capacity, reaching more than 50 % w/w, which is more than most inorganic materials and more than that usually found for organic ones. It can improve the drug loading capacity of alginate but the main effect should be the higher retention of the drug, at least at high LDH concentrations.

2. In Fig 1, FTIR and XRD of LDH  should be added as the control. Peaks of LDH/Ibu/Nap should be marked.

These FTIR and XRD have been included in the revised version

3. The section of dispersion by sonication/thermal treatment does not seem to fit in the logic of the manuscript. I don't think they are worthy in the main text. It may be fine to put in SI though. And, what is PA? Note that all abbreviations should be clearly described at the first appearance.

This section was intended to show the treatments needed for an actual utilization of the gel in tissue engineering applications. As sterilization is mandatory, we think that the provided information is relevant.

4. DLS data are not reliable because the accuracy range of Delsa Nano C instrument can only reach 7 um, whereas your data were 10 um. This should be re-done with other proper facility.

Although the accuracy of Delsa Nano C decays at 7 µm, it is still enough to detect and differentiate the size changes observed. We have performed repetitions of the experiments to ensure consistency of the data. We are currently searching for an equipment that measures with higher precision at this size range. Unfortunately, this cannot be done in the time provided for the revision.

5. SEM images are not acceptable at such scale bar. It is hard to tell if what is shown in LDH-Nap is the sample or dust. The LDH-Ibu image does not provide any valid information. They should be re-taken with better sample preparation and higher magnification with image of pure LDH as the control.

We have added additional SEM images at larger magnifications. LDH-Cl cannot be used as a control as its morphology, as explained in the text, is quite different due to the different nature of the intercalated anions.

6. The formulation used for tissue engineering is drug-loaded LDH/alg, then the drug-loaded LDH/alg should be characterised with all above techniques as well, which are missing at the moment.

We have performed several of these techniques on the mixtures, but they do not add to the overall description of the systems. In the best of cases, they are mixtures of the materials. Also, some of the samples after drug release have been characterized, but the obtained data were quite irrelevant to the obtained conclusions and they would extend the manuscript unnecessarily.

7. TEM is a "compulsory" tool when you study LDH and should be added.

There are plenty of articles that use exclusively SEM for morphological characterization. Nevertheless, we are currently looking for access to this technique, but it cannot be achieved in such a short time.

8. Sustained drug release and prolonged drug retention time are essential for tissue regeneration, the author should provide data of drug release efficiency with longer time, like 48-72 hours.

Regrettably, the time of equipment usage in shared laboratories is quite precious. Nevertheless, we think that given the clear results obtained, the main mechanisms of drug release have been already presented. On the other hand, we are pointing to perform in vivo experiments soon, which will be much more representative than in vitro release to evaluate tissue engineering applications.

9. How long can the drugs reside in the tissue for regeneration?

As mentioned before, in vivo experiments will be key to better understand the biological performance of developed systems. We believe that providing residence times based on in vitro drug release experiments would be speculative. Nevertheless, out of record, we believe that within a week would be a first approximation.

Round 2

Reviewer 1 Report

The authors have answered my comments point-by-point, and the manuscript has been greatly improved, I recommend for adoption in its present form.