Encapsulation of Ciprofloxacin into a Cyclodextrin Polymer Matrix: The Complex Formation with Human Serum Albumin and In Vitro Studies

Round 1

Reviewer 1 Report

Review of Encapsulation of Ciprofloxacin into Cyclodextrin Polymer Matrix: The Complex Formation with Human Serum Albumin and In Vitro Studies by Kopnova et al.

The manuscript entitled “Encapsulation of Ciprofloxacin into Cyclodextrin Polymer Matrix: The Complex Formation with Human Serum Albumin and In Vitro Studies” by Kopnova, Skuredina, Belogurova, and Kudryashova describes a drug formulation involving ciprofloxacin antibiotic encapsulated in a polymer matrix comprising methyl-beta-cyclodextrin-based monomers, and in vitro studies to characterize drug release against two different strains of bacteria.

The first thing this reviewer noticed about the manuscript is that it is replete with errors involving incorrect use of the English language. For example, the abstract is very confusing due to poor and unclear grammar.

o    “we propose imprinted drug ciprofloxacin (CF) into the polymer based on methyl-β-cyclodextrin (MCD)” does not make sense

o   “The obtained polycarbamide nanoparticles possess CF's sustained drug release.” This statement implies that the nanoparticles have the same properties as CF itself.

o   “The interaction of human serum albumin (HSA) with CF and CF-MCD carriers was conducted by FRET…” and interaction cannot be conducted by an analytical technique. Replace “conducted” with “analyzed” or “characterized” or another more appropriate word.

o   “…in the presence of MCD-based carries.” Presumably “carries” should be changed to “carriers”

o   Too many acronyms. MAX and MIC are not even defined.

In the introduction, a noun is missing between “among” and “with” on lines 25-26. On line 28 “regimen” should be “regimens”, assuming not all patients received the same exact treatments all over the world. On line 35 the word “numberless” is a strange choice and surely untrue, while “ideal” is surrounded by unusual and unnecessary brackets (this kind of distracting notation is repeated on lines 56, 58, 122, 206). On line 37 there is an inappropriate use of a semicolon.

At this point, the reviewer is unable to provide a comprehensive list of errors involving grammar, syntax, subject-verb agreement, etc., because they appear on almost every line of the manuscript. The manuscript reads as if it is a first draft or as if the authors do not have a strong command of the English language. These problematic sentence structures make the manuscript difficult and frustrating to read, and lead the reader to distrust the scientific content based on the poor composition of the writing. Almost 80% of the sentences are very poorly constructed. Therefore, it is highly recommended that the authors have someone edit the manuscript heavily to correct the many mistakes that are present from beginning to end.  

Another serious problem with the manuscript before the introduction is even finished involves a lack of rigorous scholarship. For example:

·       “Therefore, the most urgent task of pharmacology is the design new efficient drug 31 delivery systems to improve the properties of existed bioactive molecules.” This statement is an opinion and judgment on the part of the authors that is not well justified. It is recommended that statements like “most urgent” be removed. One could argue that other tasks are more urgent in pharmacology than new drug delivery systems.

·       On lines 37-41, the authors cite many side effects of CF without citing any references whatsoever.

An important note about chemical structure and reactivity: the structure of MCD as drawn in Figure 1 is misleading because it implies that beta-CD is permethylated and therefore has no free hydroxyl groups capable of reacting with the hexamethylene diisocyanate crosslinker. The cartoon of the MAX formula in the same figure does not capture the carbonate bond that would form only at the unmethylated sites on beta-CD. Furthermore, the CF molecule has a secondary amine which is much more nucleophilic than the hydroxyl groups of beta-CD, therefore it would be expected to react more rapidly with the isocyanate than the cyclodextrin would. The authors’ polymer most likely has many CF molecules attached to the hexamethylene cross-linkers at the piperazine moiety of CF. This concern is not addressed at all in the manuscript. This issue may explain why the MAX system appears not to interact with HSA. The CF molecule will not be useful as a drug if it never becomes bioavailable due to its covalent modification with hexamethylene diisocyanate. Release profiles in Figure 8 are expressed as percentages, which occludes the total release of CF and makes this affect difficult to assess based on the data.

The MAX system is particulate in nature. Are the nanoparticles, microparticles, macroscopic particles? The authors should provide size characterization data of the particles as it would presumably impact many of the system’s properties.  

Most of the experiments appear to have been conducted appropriately, though it is difficult to be certain based on the English problems. Owing to the concerns mentioned above, this manuscript is not recommended for publication unless it undergoes major revisions.

Problems with English are abundant and discussed in the comments and suggestions above. 

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Reviewer’s report

1. Original submission

1.1. Recommendation

Major revision

2. Comments to Author:

Manuscript ID: chemistry-2469234

Title: Encapsulation of ciprofloxacin into cyclodextrin polymer matrix: The complex formation with human serum albumin and in vitro studies.

Overview and general recommendation

The present study evaluates the incorporation of drug, ciprofloxacin onto methyl-β-cyclodextrin (MCD) via template synthesis, and the authors have provided an extensive analysis of the interaction of the new formulation with human serum albumin, which is imperative for any new pharmaceutical formulations. However, the present study lacks a lot of important aspects, such as the failure to discuss the research findings and their significance, which cast doubt in whether the manuscript is able to attract interest when it is published in its present form. Moderate English changes are required, especially in the terms used throughout and some grammatical errors. Concern is also raised on the significance of the present study as authors did not provide an adequate research background to justify why the present study was conducted. It is also exacerbated by how the methodology section was written, as I found some very important points were inadequate or completely missing. Therefore, I recommend that a major revision is warranted. Please find below my specific comments on the manuscript.

2.1. Major comments

1. The main concern I have is the significance of the present study. Authors should emphasize on the importance of monitoring the interaction of the formulation with HSA.

2. Authors did not provide detailed description on the method used.

3. The data presented need to be discussed comprehensively and thoroughly to showcase the significance of the present study.

2.2 Specific comments

1. Abstract

• Why was SARS-Cov-2 mentioned in the research background, when the present study did not investigate or evaluate the formulation for SARS-Cov-2, but instead the anti-microbial effects were evaluated on E. coli and β. subtilis.

• Authors did not include any research background on the importance of studying the interaction of the formulation with HSA. Authors should also emphasize on the in vitro study.

• Please provide a more concrete conclusion to conclude the abstract.

• Line 12: “was conducted” should be changed to was observed through….

• Line 13: “These analyses ….”

Line 14: “….that MCD decreases CF’s binding efficiency by 2 times, whereas CF’s encapsulation in polymer matrix doubles the Ka value.”

The claim made was in reference to what? Please specify.

• What is MAX? Please spell it in full at first mention.

2. Introduction

• Line 26: “…especially threatening among people with compromised immunity.”

• Line 29: “…to design new forms of drug.”

• Line 30: “Currently, the development of novel drugs and their clinical trials takes a long time and requires expensive research.”

• Line 31: “Therefore, the most urgent task of pharmacology is the design of new efficient drug delivery system to improve the properties of existing bioactive molecules.”

• Line 35: “Ciprofloxacin (CF) demonstrates numerous properties of ideal….”

• Line 37: Please add in relevant reference.

• Line 40: “…., sleep?” Please explain what does it mean by sleep problem?

• Line 48: “…derivatives as they are approved by FDA and proved to be effective drug carriers.” Please provide relevant references.

• Line 52: “CF complexes with …..”

• Line 56: “….by template synthesis….”

• Line 58: “The template imprinting….”

• Line 60: “Encapsulation of the drug by this method….”

• Line 67: “We also study the interactions of CF drug forms …..”

• Line 71-72: Check the sentence structure and rephrase accordingly.

3. Materials and methods

• Line 80-81: This should be incorporated in the previous paragraph. “…..are from the VKPM Kurchatov Institute (Moscow, Russia).”

• Line 86: “Synthesis of MAX system via crosslinking MCD-CF complexes.” Please define MAX.

• Line 87: Why 1320 μL? Please justify. … (1:1 mol)

• Line 90: “…HMI: monomer maintained 1?” What do authors mean by this? Please elaborate.

• Line 95-96: “….(the percentage of MCD included in the MAX particles in relation to the added during synthesis) was….”

Please explain more and rephrase.

• Line 102: Please define certain amount.

• Line 102-104: Amount of chemicals/compounds used is not stated. Please amend accordingly.

• Line 104: “…. was maintained at 0.06 mM, …”

• Line 106: Define gentle shaking

• Line 107: “…and fluorescence spectroscopy analyses, the solutions were diluted three times directly before the spectra registration.” Please justify.

• Line 122: Be consistent with how authors write the brand and manufacturer origin (state, country) or (country) only. …from Jasco (Tokyo, Japan)….

• Line 129: Check the manufacturer.

• Line 138: “….was maintained at 0.02 mM….”

• Line 142: “The equation includes ? and F,…..” Please amend accordingly.

• Line 153: Please define gentle shaking.

• Line 164: “The calculation of the binding constants …..”

• Line 166: What were the concentrations used for HSA? Please provide more explanation.

• Line 173: Why Trp-214? Please justify. Please check on the formatting for lines 173-174.

• Release kinetics studies: “1 mL of the sample was placed in dialysis bag MWCO of 3.5 kDA that was immersed in 1 mL of phosphate buffer (pH 7.4).”

Why authors chose to use dialysis bag with MCWO of 3.5 kDa? Please justify. Drug release studies also require sink condition. How did authors make sure that sink condition was achieved when only 1 mL of release media was employed in the present study. Please define the concentration of PBS used for drug release study.

• Please provide relevant reference for in vitro studies. Why overnight culture was used?

• Line 193: What did authors mean by “….on Petri dishes 4 wells were cut?” Please amend accordingly.

• Why these strains from ATCC were used? Please justify.

• Line 197: “The diameters of inhibition zones….”

• Please provide relevant reference for Equation 10.

4. Results and discussion

• Line 206: “….they can adsorb guest molecules….”

• Line 216: “….of different CF forms…”

• Line 221: “…as in free MCD, but the band’s shoulder appears.”

What did authors mean by this. Please provide more information.

• Line 224: “…low-intensity bands:….”

• Line 226: “…due to the hydrolysis...”

• Line 229-230: Does this mean that loading of CF is low? Please explain. Could it be due to the band was hindered due to the loading of CF in MCD?

• Line 231: “…(the percentage of MCD included in the MAX particles in relation to the one added during synthesis).”

Please simplify this statement as it is confusing and not adequately comprehensive.

• Line 232: “…was ca.” Please spell it in full.

• Line 233-234: “The encapsulation of CF was 0.6 μg/1 mg of MAX.”

Please represent this value in percentage, and authors need to discuss comprehensively whether this is considered as low, high or something that authors would anticipate. Please discuss more on this.

• Line 236: “…(CF prefers the organic phase instead of MCD tori).”

What did authors mean by this? Please explain.

• Line 262: “….wavelength of 280 nm, the individual CF and HSA demonstrate fluorescence emission peaks at 415 and 340 nm, correspondingly.”

• Figure 2: The spectra are not well presented. It is difficult to distinguish between peaks and compounds. Please amend accordingly.

• Figure 2a and 2b need to cited in the text.

• Line 272-273: How did authors come to this conclusion? Please justify.

• Line 274: Check the formatting how references should be inserted in text. Please refer to [34, 35].

• Line 275: “….does not affect the intensity…”

• Line 277-280: How does this take place? Why are they competing? Are these hydrophobic interactions. Please explain and discuss comprehensively.

• Line 286: “….might induce the quenching of ….”

• Line 287: “As MAX may interact only with the surface of HSA, no significant….”

• Figure 3a and 3b need to be cited in text accordingly.

• Line 288: “…of HSA’s emission does not change.”

• Line 292: ca. Please spell in full.

• Line 318: “Interestingly, for MAX….”

• Line 319-321: Please provide relevant reference and discuss more.

• Figure 4: Check on the formatting and labelling of the figure.

• Line 331: “In ternary systems, we also….”

• Line 332: “…with the increasing of HSA concentration.”

• Line 333: “But for the ternary system, the highest….”

• Line 334: Please check this sentence for any grammatical errors and spelling mistakes. Plato?

• Line 351: “The parameters correspond….”

• Line 352-355: Please provide a more comprehensive discussion and relevant references.

• Was Figure 5 cited in the text? If not, please do so.

• Please discuss more on the aggregation observed as a result of interaction with HSA. Why is it imperative?

• Line 394: “…in FTIR spectra, characteristic peaks could be observed….”

• Line 399: “….how CF and CF incapsulated….”

Please check and amend accordingly.

• Line 407: “CD’s carries….”

Please check and amend accordingly.

• Figure 7a and 7b need to be cited in text accordingly.

• Figure caption for figure 7 needs to be checked and corrected accordingly. What does C represent?

• Line 430-431: “The dialysis membrane’s pore diameter was chosen as 3.5 kDA, so only CF can be released.”

On what basis this claim was made? Please provide more information on this and relevant references to support claim made by authors.

• Line 438: Please check the formatting on inserting references in text [11]. Please amend accordingly.

• Line 438-440: “It is important to note that in [11], the drug release from the complex at pH 4.0 (when complex is more stable) leads to more significant decrease of the CF’s release rate.”

Why is this so? Why is the complex more stable at pH 4.0? Please provide a more comprehensive discussion, supported by relevant references.

• Line 442-443: The noticeable decrease observed, was this significant? Please justify.

• Line 445: “Thus, MAX demonstrates the prolonged drug release.”

Drug release study was only conducted up to 500 mins, can this be considered as prolonged drug release? Please justify and provide relevant references.

• Line 450-451: Authors used tenfold excess of HSA to model in vitro physiological conditions, please provide relevant reference.

• Line 456-460: Please provide relevant references. Check the spelling mistake of plato throughout the manuscript.

• Line 461: Rephrase and justify how did authors come to this conclusion.

• Line 462: “carries”. Please check and amend accordingly.

• Line 463: “Slow-release of CF from MAX and formation of the protein crown may change the antibacterial properties of CF in MAX.”

• Line 463-464: What is the change that authors are referring to? Please elaborate and provide relevant references.

• Line 469: “… by the rapid and reliable agar-well diffusion test.”

Please provide relevant references for this claim.

• Section 3.5: Discuss more on this. Authors did not explain the significance of the data obtained. Why increase in MIC was observed and whether this is favourable? Please discuss comprehensively. Did the authors include conventional antibiotic as control. Please justify.

5. Conclusion

Please rephrase the conclusion. Conclusion should reflect all the findings obtained from the present study. Authors must check for any grammar or spelling mistakes

6. References

Authors should check on the formatting, and be consistent throughout. Appropriate references should be included accordingly. Please refer to comments on previous sections.

Moderate English Language editing is required. Authors are advised to check the manuscript thoroughly for any grammatical errors and spelling mistakes

Author Response

Please see the attachment

Author Response File: Author Response.pdf