Development of Eudragit^® Nanoparticles for Intranasal Drug Delivery: Preliminary Technological and Toxicological Evaluation

Round 1

Reviewer 1 Report

Dear Editor,

This is a well-written article that propose a new nanoparticle technology for intranasal drug delivery. This new technology uses bioadhesive polymers aiming to explore its mucoadhesive properties improving drug absorption. They used Quasi-emulsion Solvent Evaporation methodology for the synthesis of Eudragit® nanoparticles, those polymer NP were loaded with diclofenac acid (DIC) or its epolamine salt (DIEP) for the delivery drug tests. The article presents a good technological and toxicological evaluation of NPs in terms of synthesis, stabilization of the NPs, Mucoadhesion studies, solubility studies in simulated nasal fluid and toxicity studies on mice ciliary tissue. The authors concluded that the Eudragit® nanoparticles as synthesized presented a good physico-chemical characteristic and were suitable for the administration of drugs via nasal route.

General comments

The paper is well-structured and relevant in the field since try to surpass some drawbacks in the intranasal (IN) administration of drugs in clinical condition. The introduction offers a useful overview of current research presenting a clear research question and how they intend to resolve it.

The results and discussion were presented clearly and accurately matching the methods and presenting all relevant data for supporting their discussion and final conclusions. However, there are some result that this reviewer think need some attention but is not a major problem in the manuscript.

Specific comments

L94 – How the authors could measure the release of the drug by the polymer NPs? They suggested that the drug was inside/outside of the NPs but nothing is discussed about drug delivery or referenced to. Just out of curiosity, there is any evidence or experiment that the authors did that shows that the drug is liberated over time and not all at once?

L99 – please specify what the authors means by “extremely small size”.

L257 – “homogeneous formulation with a mean size < 300 nm […]”.  The authors should be careful with the discussion about NPs homogeneities throughout the manuscript. Some time they refer only to size (without mention standard deviation) and some time to the polydispersity. It should be clear that they are complementary information.

L263 – How the authors explain the decrease in size for EUD3A-DIC4 compared to EUD3A? I would expect an increase since DIC have been incorporated.

L317 – “EUD3A_DIEP is characterized by a negative charge, probably due to the partial absorption of the drug on particles surface”. This possibility is really intriguing because both DIC and DIEP are similar molecule in terms of volume, size, and chemical structure. The authors have any insight about why this happened for DIEP? I believe that this could help to explain/support the previous question.

L332,333 - The authors should specify the NP nature since not all NP will have this behavior.

Figure 2 – The legend is missing.

L365 – “This phenomenon induces particles aggregation that can be detected as an increase in UV absorbance”. Aggregation leads not only to an absorbance increase but also an absorbance shift. Since the author are obtaining absorbance values at 650 nm would be UV-VIS absorbance not only UV absorbance.

L417 – “which is probably because the drug was completely encapsulated in the NP matrix”.  This quote contradicts previous affirmations that the DIEP (EUD3A_DIEP3) were outside of the NPs contributing to a negative zeta potential.

Author Response

Referee 1

Comments and Suggestions for Authors

Dear Editor,

This is a well-written article that propose a new nanoparticle technology for intranasal drug delivery. This new technology uses bioadhesive polymers aiming to explore its mucoadhesive properties improving drug absorption. They used Quasi-emulsion Solvent Evaporation methodology for the synthesis of Eudragit® nanoparticles, those polymer NP were loaded with diclofenac acid (DIC) or its epolamine salt (DIEP) for the delivery drug tests. The article presents a good technological and toxicological evaluation of NPs in terms of synthesis, stabilization of the NPs, Mucoadhesion studies, solubility studies in simulated nasal fluid and toxicity studies on mice ciliary tissue. The authors concluded that the Eudragit® nanoparticles as synthesized presented a good physico-chemical characteristic and were suitable for the administration of drugs via nasal route.

General comments

The paper is well-structured and relevant in the field since try to surpass some drawbacks in the intranasal (IN) administration of drugs in clinical condition. The introduction offers a useful overview of current research presenting a clear research question and how they intend to resolve it.

The results and discussion were presented clearly and accurately matching the methods and presenting all relevant data for supporting their discussion and final conclusions. However, there are some result that this reviewer think need some attention but is not a major problem in the manuscript.

Specific comments

Q1:L94 – How the authors could measure the release of the drug by the polymer NPs? They suggested that the drug was inside/outside of the NPs but nothing is discussed about drug delivery or referenced to. Just out of curiosity, there is any evidence or experiment that the authors did that shows that the drug is liberated over time and not all at once?

R1: This is an interesting point, in vitro release studies are under consideration prior to move to further in vivo investigations. For these preliminary tests, we supposed that ~60-70% of the drug was released within 24 hours basing on data available in literature in which particles were obtained with the same resins.

e.g.

DOI: 10.2174/2211738504666160906144633

DOI:10.35333/jrp.2020.116

Q2: L99 – please specify what the authors means by “extremely small size”.

R2: Following referees’ comment, the term “extremely” was removed and the sentence was re-written (red color) to specify how particles size may affect their transport after intranasal administration as follow:

“An extremely small size of NPs, in fact, Particles with size less than that of the olfactory axons could drive the release of the encapsulated drugs towards the N2B pathway [29, 30], instead than promoting their permanence on the nasal mucosa and drug systemic adsorption [31].”

Q3: L257 – “homogeneous formulation with a mean size < 300 nm […]”.  The authors should be careful with the discussion about NPs homogeneities throughout the manuscript. Some time they refer only to size (without mention standard deviation) and some time to the polydispersity. It should be clear that they are complementary information.

R3: We agree with referees’ comment, we referred to homogeneous formulation (line 257) based on the PdI value reported in Table 1. In order to help the reader, PdI value was inserted in the text (red color). According with referee, SD and PdI values were included also in other part of the manuscript to support our discussions (line 277; line 280-281).

Q4: L263 – How the authors explain the decrease in size for EUD3A-DIC4 compared to EUD3A? I would expect an increase since DIC have been incorporated.

Q5: L317 – “EUD3A_DIEP is characterized by a negative charge, probably due to the partial absorption of the drug on particles surface”. This possibility is really intriguing because both DIC and DIEP are similar molecule in terms of volume, size, and chemical structure. The authors have any insight about why this happened for DIEP? I believe that this could help to explain/support the previous question.

R4-R5: Particle size after DIC loading (EUD3A-DIC4) remained almost unchanged, there was just a slight reduction. This could be due to different possible phenomena such as particles shrinkage due to density changes. Moreover, we supposed that the drug was confined inside the system as revealed by the surface charge typical of the copolymers. On the contrary, some physico-chemical interaction occurred when the drug was encapsulated in the salt form (DIEP), since the variation in the zeta potential value and the higher diameter obtained suggested the presence of the drug absorbed on particle surface. We thank referee for its relevant observation that deserves to be deepened with additional experiments in forthcoming studyies.

Q6: L332,333 - The authors should specify the NP nature since not all NP will have this behavior.

R6: The nature of the NPs was specified as suggested.

Q7: Figure 2 – The legend is missing.

R7: We thank referee for this oversight, the legend was included in figure 2 as highlighted in red color.

Q8: L365 – “This phenomenon induces particles aggregation that can be detected as an increase in UV absorbance”. Aggregation leads not only to an absorbance increase but also an absorbance shift. Since the author are obtaining absorbance values at 650 nm would be UV-VIS absorbance not only UV absorbance.

R8: According with referee UV absorbance was corrected with UV-VIS absorbance.

Q9: L417 – “which is probably because the drug was completely encapsulated in the NP matrix”.  This quote contradicts previous affirmations that the DIEP (EUD3A_DIEP3) were outside of the NPs contributing to a negative zeta potential.

R9: According with referees’ comment, this sentence was modified in the manuscript (red color) as reported below:

“No characteristic peak of both drugs was observed in the DSC runs of EUD3A_DIC4 and EUD3A_DIEP3 formulations, which is probably because the drug was completely encapsulated in the NP matrix, as described in the literature and the amount absorbed on the surface was not detectable [49].”

Reviewer 2 Report

Manuscript ID: applsci-1600769
Title: Development of Eudragit® nanoparticles for intranasal drug delivery: preliminary technological and toxicological evaluation
by 
Roberta Corsaro, Rosamaria Lombardo, Carla Ghelardini, Lorenzo Di Cesare Mannelli, Daniele Bani, Angela Bonaccorso, Rosario Pignatello

Comments
The manuscript reports a detailed study about the promising strategic approach of intranasal administration of non-steroidal anti-inflammatory drugs, dispersed in colloidal system by a positively charged polymeric resin. The authors specifically focus on two common anti-inflammatory and pain mitigating drugs, i.e., diclofenac and diclofenac epolamine). 
The manuscript is a novel investigation, continuing a series of articles known in the literature about the validity of the intranasal administration alternative to oral ingestion. The intent of the presented experimental investigation is the assessing of the optimized formulation conditions for the delivery of colloidal systems composed with a drug and a suitable copolymer.
The “Introduction” section of the manuscript presents an updated review of the state-of-the-art knowledge about the recently proposed intranasal therapy. Literature on this item is properly referenced. The "Introduction" paragraph immediately attracts the attention by scientists and engineers involved in new formulation of stable colloidal systems, to be utilized for pharmaceutical products.
In “Materials and Methods” section, the authors report detailed information on the following items: purchasing sources, solubility procedure, preparation and characterization of nanoparticles, muco-adhesive evaluation, stability evaluation, differential scanning calorimetry, toxicological study, tissue preparation, statistical analysis. Actually the experimental investigations are conducted with well-established procedures and measurement methods, granting reliable results.
In the “Results and discussion” section, the authors report a large amount of experimental observations. The experimental findings are illustrated by two tables, four plots of the measured properties and an image showing histological features. The observed phenomenology is adequately discussed. 
The large amount of reported experimental observations is the force of the article. Specifically, the most valuable force aspect of the manuscript is the satisfactory perspective concerning the presented formulation as a suitable nasal drug administration.
The structure and the content of the manuscript are well systematized. The scientific terminology is correct. The figures are useful for supporting the presented experimental findings. The quality of the figures is good. The references are updates. The English language is easily comprehensible (I noted  a repetition “resins resins” at line 483 and a misprint “did not shown” at lines 488-489, correct “did not show”. The abstract text definitely indicates to the scientific community the research scope, the experimental findings and the relevant concluding account.
I suggest that this manuscript should be accepted for publication in the present form.

Author Response

Referee 2

Comments and Suggestions for Authors

Manuscript ID: applsci-1600769
Title: Development of Eudragit® nanoparticles for intranasal drug delivery: preliminary technological and toxicological evaluation
by Roberta Corsaro, Rosamaria Lombardo, Carla Ghelardini, Lorenzo Di Cesare Mannelli, Daniele Bani, Angela Bonaccorso, Rosario Pignatello

Comments
The manuscript reports a detailed study about the promising strategic approach of intranasal administration of non-steroidal anti-inflammatory drugs, dispersed in colloidal system by a positively charged polymeric resin. The authors specifically focus on two common anti-inflammatory and pain mitigating drugs, i.e., diclofenac and diclofenac epolamine). 
The manuscript is a novel investigation, continuing a series of articles known in the literature about the validity of the intranasal administration alternative to oral ingestion. The intent of the presented experimental investigation is the assessing of the optimized formulation conditions for the delivery of colloidal systems composed with a drug and a suitable copolymer.
The “Introduction” section of the manuscript presents an updated review of the state-of-the-art knowledge about the recently proposed intranasal therapy. Literature on this item is properly referenced. The "Introduction" paragraph immediately attracts the attention by scientists and engineers involved in new formulation of stable colloidal systems, to be utilized for pharmaceutical products.
In “Materials and Methods” section, the authors report detailed information on the following items: purchasing sources, solubility procedure, preparation and characterization of nanoparticles, muco-adhesive evaluation, stability evaluation, differential scanning calorimetry, toxicological study, tissue preparation, statistical analysis. Actually the experimental investigations are conducted with well-established procedures and measurement methods, granting reliable results.
In the “Results and discussion” section, the authors report a large amount of experimental observations. The experimental findings are illustrated by two tables, four plots of the measured properties and an image showing histological features. The observed phenomenology is adequately discussed. 
The large amount of reported experimental observations is the force of the article. Specifically, the most valuable force aspect of the manuscript is the satisfactory perspective concerning the presented formulation as a suitable nasal drug administration.
The structure and the content of the manuscript are well systematized. The scientific terminology is correct. The figures are useful for supporting the presented experimental findings. The quality of the figures is good. The references are updates. The English language is easily comprehensible                          (I noted  a repetition “resins resins” at line 483 and a misprint “did not shown” at lines 488-489, correct “did not show”. The abstract text definitely indicates to the scientific community the research scope, the experimental findings and the relevant concluding account.
I suggest that this manuscript should be accepted for publication in the present form.

R: We thank referee for its careful revision and according with its suggestions, the repetition (line 483) was removed and the correction (line 488-489) was performed.