Reviewer #1: The statistical analysis is made by the paired sample T-test in the section of materials and methods, then there are many groups design in results, such as nanoparticle size, PDI, and encapsulation efficiency, furthermore, different treatments and levels in laboratory animals' assay. So, this is one uncertain concern for author to explain in this manuscript.

First for nanoparticle size, PDI, and encapsulation efficiency results were carried out as triplicate and data represented as mean ± SD (n=3) without using the paired sample T-test.

Second for other analyses concerning the animal design two main comparisons were carried out using the paired sample T-test as follows:

The different treatments compared to induced group (represented by *).

The different treatments and induced group compared to mock treated group (represented by #) since no significant change was observed in (saline + PEG, IP-Np and IP-Np-T80) control groups versus mock-treated group so mock treated group was used as a general control.

Data represented as mean ± SD and p value is statistically significant at (***p≤0.001, **p≤0.01, *p≤ 0.05) compared to induced group and (#p≤ 0.05) compared to mock-treated group.

Reviewer #2: In the manuscript by Yassa et al, the authors formulate and evaluate ipriflavone loaded albumin nanoparticles and to ascertain their effectiveness along with free ipriflavone against lipopolysaccharide. This manuscript have provided us same evidence of drug efficacy of ipriflavone nanoparticles, but this manuscript still lack some important description and information. Please find points of criticism below.

1. The resolution of the picture is too low, such as TEM picture in Figure 1, western blot imaging in figure 6,7.

Corrected

All the figures were adjusted and modified using Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool.

2. For the Table 3, please give some discussion on how the concentrations of ipriflavone affect the size of nanoparticles particles.

Nanoparticle size

It was reported that solvent, drug to polymer ratio, surfactant concentration, organic/aqueous phase volume ratio and stirring speed influence nanoparticle size significantly whereas drug loading and entrapment efficiency were significantly influenced by solvents and drug to polymer ratio. Studies also demonstrated high drug concentration increases the loading efficiency as well as the particle size.

Effect of drug: polymer ratio on nanoparticle size

When the particle sizes were examined, it was seen that the particle size increases with the decreasing polymer amount. As some researchers pointed out, particle sizes were also observed to be proportional with dispersed phase viscosities.

References

1) Dora CP, Singh SK, Kumar S, Datusalia AK, Deep A. Development and characterization of nanoparticles of glibenclamide by solvent displacement method. Acta pol pharm. 2010;67(3):283-90.

2) Pongpaibul Y, Whitworth C. Preparation and in vitro dissolution characteristics of propranolol microcapsules. International journal of pharmaceutics. 1986;33(1-3):243-8.

3) Malamataris S, Avgerinos A. Controlled release indomethacin microspheres prepared by using an emulsion solvent-diffusion technique. International journal of pharmaceutics. 1990;62(2-3):105-11.

4) Chiao C, Price J. Formulation, preparation and dissolution characteristics of propranolol hydrochloride microspheres. Journal of microencapsulation. 1994;11(2):153-9.

3. For the “In vitro release of ipriflavone” experiment, simulated intestinal fluid and simulated gastric fluid were applied. We suggest that the drug release test should be performed using plasma/serum or cerebrospinal fluid in vitro.

In vitro release of ipriflavone experiment was carried out at two dissolution mediums pH 2 which simulated gastric fluid and pH 7.4 which simulated intestinal fluid and is applied for simulated blood fluid as it has the same pH 7.4 for simulated intestinal fluid so simulated blood fluid (SBF) was added.

4. The distribution of ipriflavone nanoparticles should be investigated.

Our study plan aimed for investigation of effect of ipriflavone along with its nanoformulation on brain so related indices including neuroinflammatory markers, APP processing, Amyloid precursor protein (APP), β-secretase (BACE1), α-secretase (ADAM-10 & ADAM-17), Amyloid β (Aβ), insulin degrading enzyme (IDE) and the effect of the treatments on this markers were assessed in brain tissue for this purpose. Other nanoparticle distribution can be carried out in our extended research.

5. We advise that the data in Table5 can be presented in the form of pictures.

Figures were presented as per your request named figure 3 (A, B, C &D) for different parameters and the table was added to supporting information file (S2 table 1).

6. To detect the expression of neuroinflammatory biomarkers in brain tissue, in addition to using western bolts, it is recommended to also use immunohistochemistry to check the expression of related proteins in brain tissue.

This study received no fund and was personally funded so Elisa, RTPCR and western blot were used to determine the change of neuroinflammatory biomarkers and we will consider immunohistochemistry in the future in an extended study.

Reviewer #3: In this manuscript, ipriflavone loaded albumin nanoparticles (IP-Np) against lipopolysaccharide (LPS) induced neuroinflammation in rats was performed. According to authors report, the following questions need to answer:

1. polysorbate-80 modified NPs and albumin NPs have been reported widely. What is difference about yours? The novelty of the manuscript needs to retrain.

Highlights of the novelties:

• Bovine Serum albumin nanoparticles containing ipriflavone drug has not been reported previously. Hence, this study aimed to formulate and evaluate ipriflavone loaded albumin nanoparticles and to ascertain their effectiveness along with free ipriflavone against lipopolysaccharide- induced neuroinflammation in rats.

• Also ipriflavone BSA nanoparticles coated with polysorbate 80 has not been reported previously.

2. Can polysorbate-80 modified albumin nanoparticles pass the blood-brain barrier? It needs to verify.

Polysorbate 80 is not cytotoxic and does not damage the blood–brain barrier of endothelial cells, making tight junctions appear to increase permeability for brain targeting.

Many reports demonstrated the role of polysorbate 80 on targeting brain as follows:

In a study by Wilson et al, 2014 for preparation of BSA nanoparticles for the delivery of gabapentin to the brain, the drug was administered into animals as free drug, gabapentin bound with nanoparticles, and gabapentin bound with nanoparticles coated with polysorbate 80. The polysorbate 80 coated nanoparticles increased the gabapentin concentration in the brain about 3 fold in comparison with the free drug.

Another study Poly(butylcyanoacrylate) nanoparticles coated with polysorbate 80 have been extensively studied for drug delivery into the brain.

It has also been reported that maximum concentration of azidothymidine was found in the brain after 1 h of administration of azidothymidine bound to nanoparticles coated with polysorbate 80.

This was also mentioned in introduction section page 4 (green highlighted) describing polysorbate 80 targeting the brain.

References

1) Wilson B, Lavanya Y, Priyadarshini S, Ramasamy M, Jenita JL. Albumin nanoparticles for the delivery of gabapentin: preparation, characterization and pharmacodynamic studies. International journal of pharmaceutics. 2014;473(1-2):73-9

2) Tröster, S.D., Miiller, U., Kreuter, J., 1990. Modification of the body distribution of poly(methylmethacrylate) nanoparticles in rats by coating with surfactants. Int. J. Pharm. 61, 85–100.

3) Wilson, B., Samanta, M.K., Santhi, K., Sampathkumar, K.P., Paramakrishnan, N., Suresh, B., 2008. Poly(n-butylcyanoacrylate) nanoparticles coated with polysorbate 80 for the targeted delivery of rivastigmine into the brain to treat Alzheimer's disease. Brain Res. 1200, 159–168.

4) Löbenberg R, Araujo L, von Briesen H, Rodgers E, Kreuter J. Body distribution of azidothymidine bound to hexyl-cyanoacrylate nanoparticles after iv injection to rats. Journal of controlled release. 1998;50(1-3):21-30.

5) Patil GB, Surana SJ. Bio-fabrication and statistical optimization of polysorbate 80 coated chitosan nanoparticles of tapentadol hydrochloride for central antinociceptive effect: in vitro–in vivo studies. Artificial cells, nanomedicine, and biotechnology. 2017;45(3):505-14.

6) Sun W, Xie C, Wang H, Hu Y. Specific role of polysorbate 80 coating on the targeting of nanoparticles to the brain. Biomaterials. 2004;25(15):3065-71.

7) Kreuter J, Ramge P, Petrov V, Hamm S, Gelperina SE, Engelhardt B, et al. Direct evidence that polysorbate-80-coated poly (butylcyanoacrylate) nanoparticles deliver drugs to the CNS via specific mechanisms requiring prior binding of drug to the nanoparticles. Pharmaceutical research. 2003;20(3):409-16.

3. Oral administrated with ipriflavone-loaded albumin nanoparticles coated with tween 80, would albumin nanoparticles be degraded in the gastrointestinal track? If like this, what’s the meaning of nano drug delivery?

Physicochemical characteristics of nanoparticles differ than macromolecules in gastrointestinal track and studies revealed that the absorption of albumin nanoparticles in the gastrointestinal tract has an optimal particle size. Therefore, only nanoparticles with suitable particle sizes can be prepared to meet the clinical needs.

Several studies revealed the preparation of BSA nanoparticles and microspheres for oral delivery of the drugs.

Also in our study in vitro drug release test from nanoparticles using simulated gastric fluid pH 2 was presented (page 14).

References

1) Girotra P, Singh SK. A comparative study of orally delivered PBCA and ApoE coupled BSA nanoparticles for brain targeting of sumatriptan succinate in therapeutic management of migraine. Pharmaceutical Research. 2016;33(7):1682-95.

2) Shahgholian N, Rajabzadeh G, Malaekeh-Nikouei B. Preparation and evaluation of BSA-based hydrosol nanoparticles cross-linked with genipin for oral administration of poorly water-soluble curcumin. International journal of biological macromolecules. 2017;104:788-98.

3) Golla K, Reddy PS, Bhaskar C, Kondapi AK. Biocompatibility, absorption and safety of protein nanoparticle-based delivery of doxorubicin through oral administration in rats. Drug delivery. 2013;20(3-4):156-67.

4) Desai MP, Labhasetwar V, Amidon GL, Levy RJ. Gastrointestinal uptake of biodegradable microparticles: effect of particle size. Pharm Res. 1996;13:1838–45.

5) Hussain N, Jaitley V, Florence AT. Recent advances in the understanding of uptake of microparticulates across the gastrointestinal lymphatics. Adv Drug Deliv Rev. 2001;50:107–42

6) Yeboah KG, D’souza MJ. (2009). Evaluation of albumin microspheres as oral delivery system for Mycobacterium tuberculosis vaccines. J Microencapsul 26:166–179

7) Sahin S, Selek H, Ponchel G, Ercan MT, Sargon M, Hincal AA, Kas HS.(2002). Preparation, characterization and in vivo distribution of terbutaline sulfate loaded albumin microspheres. J Control Release 82:345–358.

8) Shivakumar HN, Vaka SR, Murthy SN. (2010). Albumin microspheres for oral delivery of iron. J Drug Target 18:36–44.

4. The different indexes have the similar trends of fig3,4,5 in results, therefore repeated descriptions presented.

Different parameters were used to detect the expression of neuroinflammatory biomarkers in brain tissue as TNF-α, IL-6, IL-1β, iNOS, amyloidogenic parameters (Amyloid-β & IDE) and ACHE activity where they revealed significant increase in their levels in Lps induced group and there were significant reduction in ipriflavone treated and nanoparticle formulation treated groups. Different parameters were gathered with similar interpretation.

5. In Fig4,6, western-blot results can put alone.

The figures were modified.

This manuscript by Nashwa W. Yassa et al reports a neuroprotective effect of ipriflavone-loaded nanoparticle in LPS-induced brain inflammation of adult male rats. High dose of ipriflavone is not well-suited for therapy in their previous research. BSA is a useful carrier with drug binding sites, non-immunogenic and easily biodegradable in vivo. So, it is both interesting and practical to novel drug delivery systems and current treatment of neurodegenerative diseases. This will attract interests from readers, however, there are some concerns to be made clearly.

1. In the section of Materials and methods, volume unit “ml” should be changed to be “mL” in the full manuscript.

Corrected (yellow highlighted)

2. In animal experimental model, rats’classification is designed to twelve groups. I should expect author to address this issue in discussion or provide related reference or report in previous literature. Overall, this work suggests a useful and impactful strategy for preparation of ipriflavone-loaded nanoparticle and therapeutic applications in neurodegenerative diseases.

The animal classification was discussed in page 21 in Discussion section. More explanation were added to the paragraph (yellow highlighted) and S2 fig 1 was cited showing Experimental Design for the studying of ipriflavone and its nanoparticle preparation against LPS induced neuroinflammation in rats.

3. The authors mentioned “by the anti-inflammatory, antioxidant and gene-remodeling activities” on line 583 in page 28. The gene-remodeling activities are not efficiently addressed in results, although the western blot assay and RT-PCR are performed to indicate the difference between treatment groups and plain groups.

In conclusion section this was corrected to anticholinesterase as the changes in gene expression profile was discussed in details in discussion section.

Also a schematic diagram for the effect of ipriflavone and ipriflavone nanoformulation on LPS brain inflammation induced rats (S2 Fig2) in supporting information file was cited in conclusion section.

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Additional Editor Comments (if provided):

In general, findings presented here are novel, experiments seem to be well done, and paper describes an interesting topic. However, there are some major points that complicate acceptance of the paper in the present format.

The editor makes the following suggestions:

1. Provide immunofluorescence staining and Western blot analysis of NF-kB in each group.

Since our major study focused on preparation of nanoparticle, its characterization and study of its effect using biochemical and molecular methods, the tissues were not kept in formalin for the immunofluorscence and we will consider it in the future in an extended study.

Western blot analysis for NF-kB p65 was added at figure 7 D and original blot image was added to s1- raw images file.

Also NF-KB p65 was analysed using semi-quantitative RT-PCR shown in fig 7C and original gel image is supplied in supplementary file raw images.

2. Provide staining of oxidative stress in each group.

Oxidative stress parameters (TBARS, GSH, GST, GPX, SOD AND nitric oxide production) were analyzed using biochemical methods as shown in fig.3 and data are presented in table1 in s2 file. Tissues were not kept in formalin and we will consider it in our future studies.

3. The discussion should be improved correlating these results with the literature.

A part was added yellow highlighted alongside what was mentioned before (green highlighted) correlating these results with previous literature (four references were added 79-82)

Also a schematic diagram (fig 9) was added on page 28

4. The manuscript is not linked to current conversations in the journal.

All journal requirements were fulfilled.

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