Biodistribution of Fluorescent Albumin Nanoparticles among Organs of Laboratory Animals after Intranasal and Peroral Administration

Round 1

Reviewer 1 Report

This is an interesting manuscript reporting on a nice experiment about biodistribution of fluorescent BSA nanoparticles after administration through two direct ways (intranasal and orally). The topic is of interest but several issues have to be addressed to improve the manuscript before considering it for publication, because several statements are not supported by data, probably because of incomplete data presentation more than for the unavailability of these results.

The comments are:

 

The dye is sensitive to oxidation by ROS and acidic degradation. Some organs and biological processes may have higher level of ROS and lead to a decrease of fluorescence which does not mean a lower uptake. This is a severe issue which should be discussed very clearly in the manuscript to identify the boundaries of the experimental results and their interpretation. This especially interesting for liver, spleen and kidneys where immune cells may work with acids to destroy external particles.

 

Where urin and feces also checked for fluorescence?

 

In figure 2 the histograms for oral administration are not reported. In figure 3, the colours are too similar, each label should be linked to cake slice with a line and not in a separate legend.

 

It is unclear if the penetration in mouse brain is homogeneous or any gradient was observed and which regions were more fluorescent.

 

Immunogenicity is mentioned but there are no results presented about it, so it should be removed from claims of the manuscript. For instance in conclusions “Innate immune response in mice was weak (if any).” There are no data about it, this couldn’t be stated.

 

In conclusions: “with peak values in 1-2 days and subsequent decline”: there are no data, neither qualitative fluorescence, showing this decline or what happens after 2 days.

 

There are no images of fluorescence in other organs after oral administration.

 

 

The introduction provides a wide overview of the proteins and their role in nanomedicine, yet a relevant aspect has not been mentioned, concerning the coating of inorganic biodegradable nanomedicine as those described in Journal of Colloid and Interface Science 596 (2021) 332–341; Chem. Sci., 2019, 10, 5435-5443; PNAS 2019, 117 (1) 103-113; ACS Nano 2015, 9, 8, 7925–7939; ACS Nano 2020, 14, 10, 12840–12853). It is suggested to add these references to mention that protein coatings are crucial also for the transport of these next generation inorganic nanomedicines.

 

 

language is fine

Author Response

This is an interesting manuscript reporting on a nice experiment about biodistribution of fluorescent BSA nanoparticles after administration through two direct ways (intranasal and orally). The topic is of interest but several issues have to be addressed to improve the manuscript before considering it for publication, because several statements are not supported by data, probably because of incomplete data presentation more than for the unavailability of these results.

Answer:

            We greatly appreciate your attention, research interest and valuable suggestions for improvement of our manuscript and even for further development. The text was modified accordingly and corrections are highlighted as Microsoft Word track changes for your convenience.

 

The comments are:

 

The dye is sensitive to oxidation by ROS and acidic degradation. Some organs and biological processes may have higher level of ROS and lead to a decrease of fluorescence which does not mean a lower uptake. This is a severe issue which should be discussed very clearly in the manuscript to identify the boundaries of the experimental results and their interpretation. This especially interesting for liver, spleen and kidneys where immune cells may work with acids to destroy external particles.

 Answer:

            To our knowledge fluorescent rhodamine dyes and especially RhoB are more stable compared to fluorescein derivatives and cyanine dyes. RhoB fluorescence emission does not depend on the protein concentration, pyrine nucleotides in close proximity and slight variations of pH near physiological range of pH 6-8. There are two buffers (carbonate and phosphate salts) in order to maintain pH stability in living system. Protein NP adsorption and subsequent unspecific endocytosis result in RhoB-BSA NP accumulation in lysosomes with pH~5 where proteolytic processing is known to take place. Antigen presentation with the major histocompatibility complex (MHC) I complex induces Th1 innate and adaptive immunity as was described in our previous publications [9, 18]. Further research is necessary to determine a role of ROS. Weak (if any) innate immunity suggested negligible specific cellular and humoral immunity. Consequently, the fluorescent protein NP elimination from living organisms takes place without involvement of immune system. The discussion was revised accordingly. 

Where urin and feces also checked for fluorescence?

  Answer:

Unfortunately, not. Our research was aimed at distribution of the fluorescent protein NP among organs of laboratory animals after intranasal and oral instillation but not at their elimination and excretion. It might be a direction of further research using other experimental approaches. To study dynamics of protein NP removal from living organisms special individual collectors for urine and feces are required.

 

In figure 2 the histograms for oral administration are not reported. In figure 3, the colours are too similar, each label should be linked to cake slice with a line and not in a separate legend.

Answer:

All data are available on request. Unfortunately, according to instructions to authors the number of figures in the Special Issue is limited. Our revised manuscript includes 5 multi-panel figures and each of them should be carefully discussed.  Evident observed variations between intranasal and peroral ways as well as among animals in each group are described in our revised text.

Figure 3 shows prevalent accumulation of RhoB-BSA NP in brain and intestine with minor fractions in other organs without presentation of average values and standard deviations for each organs just for visual presentation. Our attempts to link each cake slice with a line and corresponding label with visible text were not successful.

 

It is unclear if the penetration in mouse brain is homogeneous or any gradient was observed and which regions were more fluorescent.

Answer:

            Prevalent accumulation of RhoB-BSA NP in olfactory bulbs, cerebellum and cerebral cortex was not observed (Figure 5). In each field of view of each histological section of each rabbit brain part the fluorescence emission was not homogenous. One can see intracellular and extracellular RhoB probably resulted from endocytosis and exocytosis of the RhoB-BSA NP, respectively.  However, number of rabbits in each group was limited. Mouse brain parts were not isolated, identified and studied properly.

 

Immunogenicity is mentioned but there are no results presented about it, so it should be removed from claims of the manuscript. For instance in conclusions “Innate immune response in mice was weak (if any).” There are no data about it, this couldn’t be stated.

 Answer:

  Cytokine gene expression was estimated in 1 day after RhoB-BSA NP intranasal and peroral administration by means of RT²-PCR. RNA of IFN of I, II and III types, as well as interleukins IL12 and 17 were found neither in the mouse blood leukocytes nor in sera. Only IL4 and IL10 RNA were detected in one from 10 mice. The data are described in our revised text but the negative results of real time PCR were not shown on the additional figure. All data are available on request.

 

In conclusions: “with peak values in 1-2 days and subsequent decline”: there are no data, neither qualitative fluorescence, showing this decline or what happens after 2 days.

 Answer:

            The results were previously described in the text and our published articles [9] and were not shown in the overloaded figures. The statement about “subsequent decline” was removed from the revised section “Conclusion”.

 

There are no images of fluorescence in other organs after oral administration.

  Answer:

            All data are available on request. It seems to be hardly possible to show numerous images of fluorescent microscopy for 7 organs of 30 mice (10 intact, 10 after intranasal instillation, 10 after oral administration of the RhoB-BSA NP) in dynamics because of limited volume of each research paper.

 

The introduction provides a wide overview of the proteins and their role in nanomedicine, yet a relevant aspect has not been mentioned, concerning the coating of inorganic biodegradable nanomedicine as those described in Journal of Colloid and Interface Science 596 (2021) 332–341; Chem. Sci., 2019, 10, 5435-5443; PNAS 2019, 117 (1) 103-113; ACS Nano 2015, 9, 8, 7925–7939; ACS Nano 2020, 14, 10, 12840–12853). It is suggested to add these references to mention that protein coatings are crucial also for the transport of these next generation inorganic nanomedicines.

Answer:

            All suggested references were read and carefully checked.

            The first paper https://www.sciencedirect.com/science/article/pii/S0021979721003908 published in Journal of Colloid and Interface Science 596 (2021) 332–341 describes silver-iron nanoparticles coated with polyethylene glycol but not with a protein. So it seems not to be relevant to the content of our manuscript.

            The second paper https://pubmed.ncbi.nlm.nih.gov/31293725/ published in Chem. Sci., 2019, 10, 5435-5443 is about PEGylated rhenium nanoclusters and includes nothing about protein NP accumulation dynamics in organs of living organisms.

            The third article from the suggested list https://www.pnas.org/doi/10.1073/pnas.1911734116  (PNAS 2019, 117 (1) 103-113) is devoted to 2-step lifecycle of gold nanoparticles captured by primary fibroblasts in vitro but not in vivo. Protein shells are missing.

            The fourth article https://pubs.acs.org/doi/abs/10.1021/acsnano.5b00042 (ACS Nano 2015, 9, 8, 7925–7939) represents study of long-term fate of gold/iron oxide heterostructures covered with two different coating shells - amphiphilic polymer or polyethylene glycol but not with a protein after intravenous injection in mice and it appears to be also far from our research.

            The last from the list https://pubmed.ncbi.nlm.nih.gov/32877170/ published in ACS Nano 2020, 14, 10, 12840–12853 describes gold-iron alloys that are not relevant to our research.

            Introduction of our manuscript revised according to recommendations of both editor and reviewers describes natural and artificial nanostructures consisting of different organic and inorganic materials [6-12]. Bio-reactivity of different NP is commonly based on protein interactions due to biocorona formation immediately after addition of biorelevant media or administration into organisms. Therefore, protein coating is indeed crucial for the transport, biodistribution and immune response induced by NP of various chemical composition and different origin. Taking into consideration the limited volume of our research paper but not a comprehensive review all relevant references are focused on protein nanomaterials in organs of living organisms.

Author Response File: Author Response.doc

Reviewer 2 Report

The aim of the manuscript is valuable for the biomedical field, however, there are many points, both conceptual and formal, to address before its acceptance.

1.    Title. The first part is too broad since only one type o nanoparticles is described.

2.    Introduction.

a.    The interest of BSA nanoparticles in biomedicine should be noted. What is the ability of BSA Nanoparticles to retain drugs and thus be used as drug delivery systems?

b.    The last part should be better linked.

3.    Methods. Spectrofluorometry and Fluorescent microscopy are techniques for obtaining results, then the aim of the test should be the headline. To make me clear, both techniques are used to follow nanoparticles’ biodistribution.

4.   Results.

a.    Control samples are needed. Only BSA-Rho-B nanoparticles are studied. The aim of the research is to use BSA nanoparticles as drug delivery systems without the presence of Rho-B, isn’t it? Then, results should be compared to BSA Nanoparticles.

b.    Rho-B is chemically bound to BSA nanoparticles to follow their biodistribution, but BSA has native fluorescence.

c.    Figure 1. Y axes should indicate % intensity and scale bar should be shown in the images.

d.    What is swab? It is not mentioned in methods section. Why to check stability in water if the aim of the study is to use these nanoparticles in vivo.

Author Response

The aim of the manuscript is valuable for the biomedical field, however, there are many points, both conceptual and formal, to address before its acceptance.

Answer:

Thank you for your careful attention and valuable suggestions for revision of our manuscript. All your queries were addressed. New insertions in our revised manuscript are highlighted as Microsoft word track changes and additionally marked in blue color whereas deletions are shown in red font with a strikethrough as Microsoft word track changes.

 

1. The first part is too broad since only one type o nanoparticles is described.

Answer:

The title was changed to the following.

“Biodistribution of fluorescent albumin nanoparticles among organs of laboratory animals after intranasal and peroral administration”

 

2. Introduction.
3. The interest of BSA nanoparticles in biomedicine should be noted. What is the ability of BSA Nanoparticles to retain drugs and thus be used as drug delivery systems?

 

Answer:

General properties of all albumins such as stability, low immunogenicity, binding with free radicals, drugs and other harmful chemicals as well as antioxidant and anticoagulant properties are described in the revised “Introduction” whereas details specific for BSA are at the beginning of revised “Discussion” in order to avoid any repeats.

 

1. The last part should be better linked.

 

Answer:

Long-term fate of foreign nanomaterials is determined by oxidative stress, reactive oxigen species (ROS), innate resistance and adaptive immune response. Cytokine gene expression immediately after antigen presentation can regulate polarization of subsequent specific cellular and humoral response. To decrease unwanted side effects, allergic complication and autoimmine diseases NP-induced innate immunity should be studied.

The end of “Introduction” was revised accordingly.

 

 

2. Spectrofluorometry and Fluorescent microscopy are techniques for obtaining results, then the aim of the test should be the headline. To make me clear, both techniques are used to follow nanoparticles’ biodistribution.

Answer:

RhoB-BSA NP were not extracted from organs of experimental mice and rabbits. To our knowledge, it is hardly possible to isolate foreign protein nanoparticles from organs because of ubiquitous extracellular vesicules and intracellular nanoparticles of various chemical composition and different origin for targeted transport within living cells by means of physico-chemical methods.  To detect biodistribution of the pre-labelled fluorescent protein NP both quantitative spectrofluorometry and fluorescent microscopy were used and described in details in the revised manuscript. Fluorescence emission with maximum at 580 nm at excitation wavelength 550 nm that is typical for the fluorescent rhodamine dye RhoB in the protein NP and far from the autofluorescence of phenyl alanine (282 nm), tyrosine (303 nm) and tryptophane (348 nm) amino acid residues of proteins from living cells. The fluorescence was daily measured for 10% homogenates of organs of laboratory animals in PBS after intranasal and peroral administration in dynamics using spectrofluorometer “Fluoromax+” (Horiba Scientific, Japan).

For fluorescent microscopy histological sections and prints of the mouse and rabbit organs in 2 days after intranasal and peroral administration of RhoB-BSA NP on glass slides were fixed with 4% cold formaldehyde solution in PBS for 10 min at room temperature. After washes with cold PBS 3 times the cellular DNA were additionally stained with the fluorescent intercalating dye Hoechst 33342 (Abcam, USA). The fluorescent microscopy was performed using the microscope Nikon Eclipse Ti (Nikon, Japan) with a Plan Fluor 20×/0.45 objective (Nikon, Japan) with a set of filters providing excitation/emission for RhoB (528–553 nm/590–650 nm) and for Hoechst 33342 (340-380 nm/435-485 nm). The images were captured with camera ORCA-Flash4.0 (Hamamatsu, Japan) with exposure times 100-500 ms.

4. Results.
5. Control samples are needed. Only BSA-Rho-B nanoparticles are studied. The aim of the research is to use BSA nanoparticles as drug delivery systems without the presence of Rho-B, isn’t it? Then, results should be compared to BSA Nanoparticles.

 

Answer:

Unlabeled BSA nanoparticles remain undetectable inside cells and organs of animals because of numerous natural solid nanoparticles  and vesicles. Comparison of cellular uptake of the fluorescent rhodamine dye RhoB, the fluorescent protein RhoB-BSA and the fluorescent protein nanoparticles RhoB-BSA NP were earlier studied in details in dynamics and published in [9]. Further research is required for drug delivery by means of BSA nanoparticles.

 

1. Rho-B is chemically bound to BSA nanoparticles to follow their biodistribution, but BSA has native fluorescence.

 

Answer:

To our knowledge, there are not physical or chemical methods that permit to isolate artificial protein NP from natural intracellular and extracellular solid NP and vesicles. Therefore, foreign protein NP were not extracted from organs of experimental animals. In order to detect biodistribution of the pre-labelled fluorescent protein NP both quantitative spectrofluorometry and fluorescent microscopy were used and described in details in the revised manuscript. Fluorescence emission with maximum at 580 nm at excitation wavelength 550 nm is typical for the fluorescent stable rhodamine dye RhoB in the protein NP and far from the autofluorescence of phenyl alanine (282 nm), tyrosine (303 nm) and tryptophane (348 nm) amino acid residues of proteins from living cells including all albumins and, in particular, BSA.

 

1. Figure 1. Y axes should indicate % intensity and scale bar should be shown in the images.

Answer:

Figure 1 shows dynamic light scattering (DLS) data. RhoB-BSA NP hydrodynamic radii were measured by dynamic light scattering (DLS) using NANO-flex 180° (Microtrac, USA). Software of our equipment calculate “% passing” but not “% intensity” that shown in upper panel.

Scale bars were added into each image of the revised figures 1, 4 and 5. Legends of the revised figures were corrected.

 

 

1. What is swab? It is not mentioned in methods section. Why to check stability in water if the aim of the study is to use these nanoparticles in vivo.

 

Answer:

            Swab is the medical term. Commonly soft tip is gently inserted into the mouth and is rubbed in a circle near the inside of the cheek several times in order to collect mucosal layer and epithelial cells. The method is widely used in clinical laboratories. Therefore, there is no need to describe it in details.

            RhoB-BSA NP stability in water was previously analyzed to estimate nanoprecipitation as convenient method for protein NP fabrication. The data were earlier published (Morozova et al., 2018) [18].

 

Author Response File: Author Response.doc

Reviewer 3 Report

 

This paper compares the biodistribution of rhodamine labelled albumin nanoparticles in mice and rabbits after intranasal and peroral administration, as indicated by the title, and shows their selective accumulation within the brain and intestine, without significant immune reaction.

 There is, obviously, a clear interest in approaches allowing a better access of substances, i.e. drugs and therapeutic proteins, to this organ protected by the blood brain barrier.  Nevertheless, at this stage, the paper should be significantly improved to deserve publication.

     ·        The term RhoB may be confusing for cell biologists and should probably be adapted to avoid this problem.

·        A validation of the spectrofluorometric method to assay NP distribution, even briefly, should be given.

·        The RhoB / BSA ratio should be given since the fluorescent tag could affect the fate of the NP, as known for others.

·        Some details should be given on the modes of administration of the NP.

·        Figure 4: difficult to see details.  Possible to enlarge the images as in figure 5?

·        The potential presence of specific RNA in sera should be explained.

About the discussion, some items should be adapted or clarified.

·        Is such a high stability of the NP in the presence of plasma a real advantage aiming at using them for drug delivery avoiding immune reaction?

·      I do not see the real interest of the first paragraph of the discussion.

·        This section should also discuss on the accumulation of NP in the intestine.

·        A comparison with other proteins, e.g. antibodies should be discussed.

·        What about possible toxicity of these NP in the brain, due to their long stability?

·        Examples of therapeutic applications, i.e. proteins, drugs,… should be given.

 

 

Author Response

This paper compares the biodistribution of rhodamine labelled albumin nanoparticles in mice and rabbits after intranasal and peroral administration, as indicated by the title, and shows their selective accumulation within the brain and intestine, without significant immune reaction.

 There is, obviously, a clear interest in approaches allowing a better access of substances, i.e. drugs and therapeutic proteins, to this organ protected by the blood brain barrier.  Nevertheless, at this stage, the paper should be significantly improved to deserve publication.

Answer:

We would like to thank the Reviewer for the suggestions and provide a point by point replies to the comments. For your convenience all new insertions in our revised manuscript are highlighted as Microsoft word track changes and additionally marked in blue color whereas deletions are shown in red font with a strikethrough as Microsoft word track changes.

 

• The term RhoB may be confusing for cell biologists and should probably be adapted to avoid this problem.

Answer:

Fluorescent rhodamine dye RhoB was used to label the protein – the bovine serum albumin in order to construct fluorescent protein nanoparticles. To our knowledge fluorescent rhodamine dyes and especially RhoB are more stable compared to fluorescein derivatives and сyanine dyes. RhoB fluorescence emission does not depend on the protein concentrations, pyrine nucleotides in a close proximity and slight variations of pH near physiological range of pH 6¸8. Fluorescence emission with maximum at 580 nm at excitation wavelength 550 nm is typical for the fluorescent rhodamine dye RhoB and far from the autofluorescence of phenyl alanine (l max=282 nm), tyrosine (l max=303 nm) and tryptophane (l max=348 nm) amino acid residues of proteins from living cells. Consequently, that fluorescence emission spectra of the rhodamine dye RhoB and cellular proteins are not overlapping. The revised text was corrected accordingly.

• A validation of the spectrofluorometric method to assay NP distribution, even briefly, should be given.

Answer:

Spectrofluorometry with the fluorescence excitation at 550 nm and emission at 580 nm was measured for 10% homogenates of organs of mice in PBS after intranasal and peroral administration in dynamics using spectrofluorometer “Fluoromax+” (Horiba Scientific, Japan) as described in the revised section “Materials and methods”. Validation of the spectrofluorometric method was earlier published in [9, 18] with necessary controls including the fluorescent dye RhoB solution with known concentrations and molar extinction coefficient, the fluorescent labelled protein RhoB-BSA and the corresponding nanoparticles. Autofluorescence of organs of intact mice and rabbits in this range near 580 nm was minimal as shown by means of fluorescent microscopy (Figure 4).

• The RhoB / BSA ratio should be given since the fluorescent tag could affect the fate of the NP, as known for others.

Answer:

RhoB / BSA molar ratio was estimated before the labelling reaction and did not exceed 10 in order to reduce a risk of conformational changes as previously described [18]. BSA was labelled with rhodamine B (Rho B) in 0.1 M Na₂CO₃ solution at pH 9.3 for 1 h at room temperature and then was purified by Sephadex G25 gel-chromatography with mild centrifugation at 700 g [18]. Trace amounts of the free fluorescent dye RhoB cannot be excluded from Sephadex G25 eluates. Nevertheless, according to our estimations and based on the known molar concentrations of BSA and RhoB with known molar extinction coefficients RhoB dye excess was 3-5 in comparison with BSA molecules. BSA antigenic and conformational stability was confirmed by ELISA with polyclonal antibodies against BSA as earlier described in [18]. The text was revised accordingly.  

 

• Some details should be given on the modes of administration of the NP.

Answer:

Intranasal and peroral instillation of the fluorescent protein NP in mice and rabbits was described in details in the new separate section of “Material and methods” of our revised manuscript with the corresponding title.

 

• Figure 4: difficult to see details. Possible to enlarge the images as in figure 5?

Answer:

Figure 4 was revised. It is multi-panel figure with all necessary controls that cannot be removed. Scale bars were added in each image of the revised figure 4. Final size of any on-line image may be enlarged for convenience.

• The potential presence of specific RNA in sera should be explained.

Answer:

In order to detect cytokine gene expression total RNA were isolated from 100 µl of mouse blood cells and sera using “Proba-NK” kit. RNA isolation from blood sera is widely used in molecular biology, immunology and clinical diagnostics. RNA is evidently derived from destroyed cells. Usually, quantities of genome-equivalents of any specific RNA in sera did not exceed those in blood cells. However, isolation of labile RNA from blood cells is complicated by RNase-mediated hydrolysis and high concentrations of cellular proteins. Moreover, hemoglobin from erythrocytes of blood is powerful inhibitor of both MMLV reverse transcriptase and Taq DNA dependent DNA polymerase that are necessary for reverse transcription with subsequent real time PCR.

About the discussion, some items should be adapted or clarified.

• Is such a high stability of the NP in the presence of plasma a real advantage aiming at using them for drug delivery avoiding immune reaction?

Answer:

  Nanoparticles can entry into eukaryotic cells by means of unspecific endocytosis without receptor involvement. Our experiments with 3 human cell lines and human mononuclear blood cells showed that the same fluorescent protein nanoparticles RhoB-BSA NP were accumulated inside the studied cells in 2-4 hours posttreatment. The data were earlier published in [9]. So, the time period of extracellular circulation of foreign protein nanoparticles and their possible contacts with biological fluids including blood plasma and saliva did not exceed a few hours. The data were also confirmed by incubation of the RhoB-BSA NP in the presence of blood serum and mouth swabs as shown on figure 1. Our research was aimed at intracellular delivery and pharmacodynamics.

BSA and corresponding NP are known to lack immunostimulatory signals.

 

•      I do not see the real interest of the first paragraph of the discussion.

Answer:

The first paragraph of the discussion was significantly revised according to the recommendation of other reviewer. General properties of all albumins such as stability, low immunogenicity, binding with free radicals, drugs and other harmful chemicals as well as antioxidant and anticoagulant properties are described in the revised “Introduction” whereas details specific for BSA are at the beginning of revised “Discussion” in order to avoid any repeats and to begin “Discussion” with comprehensive analysis of BSA stability in water in wide ranges of pH and temperatures during NP fabrication by nanoprecipitation and subsequent long-term storage for several years.

 

• This section should also discuss on the accumulation of NP in the intestine.

Answer:

Discussion of the RhoB-BSA NP accumulation in the intestine was added with two recently published reviews [25, 26] in the references list at the end of the revised manuscript.  

• A comparison with other proteins, e.g. antibodies should be discussed.

Answer:

NP consisting of different proteins including BSA, human and mouse polyclonal and monoclonal antibodies, fibrinogen, 7 biomedicine relevant enzymes were fabricated using the same method of nanoprecipitation and previously described in [9, 18 and Morozova et al., 2023]. The solid protein NP of different sizes retained their enzymatic and ligand-binding properties [9, 18 and Morozova et al., 2023].

Morozova O.V.; Barinov N.A.; Klinov D.V. Stable Enzymatic Nanoparticles from Nucleases, Proteases, Lipase and Antioxidant Proteins with Substrate-Binding and Catalytic Properties. Int. J. Mol. Sci. 2023, 24, 3043. https://doi.org/10.3390/ijms24033043.

Our manuscript was aimed at biodistribution of the protein NP among organs of laboratory animals in vivo. Comparison of BSA NP with other protein NP was not performed because of limited number of experimental animals for simultaneous handling, processing and subsequent time-consuming analysis (fluorescent microscopy of multiples histological sections and prints).

 

• What about possible toxicity of these NP in the brain, due to their long stability?

Answer:

            Acute toxicity of the RhoB-BSA NP after intranasal and peroral instillation was not registered during period  of our observations for 7 days. Neither weight loss, nor refusal to eat or to drink, nor other morphological or behavioral disorders were observed. Further research is necessary to estimate chronic toxicity and other possible complications of the nanomaterials.

 

• Examples of therapeutic applications, i.e. proteins, drugs,… should be given.

Answer:

Examples of therapeutic applications of albumins as drug carriers have been added in the revised “Introduction”. Albumin was one of the first FDA-approved proteins because of its high thermal and pH stability, antioxidant, anticoagulant and low immunogenic properties. The commercially available albumin-based drugs include Abraxane (Celgene) (paclitaxel loaded albumin NP), optison (GE Healthcare) (human serum albumin stabilized perflutren microspheres as ultrasound contrast agent) and albumin-bound anti-cancer drugs (ABI-009 (Aadi with Celgene) - albumin bound rapamycin and ABI-011 (NantBioScience) - albumin bound thiocolchicine analog (IDN 5405)) [9 and references therein].

Author Response File: Author Response.doc

Reviewer 4 Report

In this manuscript, the authors carry out an in vivo study of the distribution of RhoB-BSA NP after intranasal and oral administration of NP in mice and rabbits, evaluating innate immunity in both cases. There are considerations to take into account:

1. The intranasal and oral route of administration is used but not the route of administration through the tail vein. This must be justified.

2. The type of laboratory animals used must appear in the abstract.

3. They use control groups in both mice and rabbits but the results obtained in them are not mentioned.

4. In the manuscript the authors say that the nanoparticles are stored in different tissues and compare day 1 and day 2. In the conclusion of this study it is said “they accumulated mainly in brain and intestine with peak values in 1-2 days and subsequent decline” lines 424-425. The results do not support this conclusion as there are no data beyond day 2.

5. The text says “Available data on stability of protein NP in vitro permit to suggest their implementation in nanomedicine as diagnostic tools and potential drugs.” lines 335-336 Reference is needed.

6. In the "fluorescent microscopy" methodology, it is necessary to specify the size of the sections obtained.

7. Figures 1, 4 and 5 need a scale bar on the images.

8. 36% plagiarism has been detected, attached document, must be corrected

Comments for author File: Comments.pdf

Author Response

In this manuscript, the authors carry out an in vivo study of the distribution of RhoB-BSA NP after intranasal and oral administration of NP in mice and rabbits, evaluating innate immunity in both cases.There are considerations to take into account:

1. The intranasal and oral route of administration is used but not the route of administration through the tail vein.This must be justified.

Answer:

Non-invasive drug delivery includes painless drug administration across the biological barriers of the mucosal surfaces or the skin. Besides induction of innate and adaptive systemic immune response both intranasal and oral instillations may cause mucosal immunity to prevent respiratory and intestinal infections. It is hardly possible to compare different administration ways in a research because of limited number of experimental animals for simultaneous handling, treatment and processing of samples.  The text of “Introduction” was properly revised.

2. The type of laboratory animals used must appear in the abstract.

Answer:

            The sentence of the Abstract was corrected to the following.

“The fluorescent protein NP were administered intranasally and orally in laboratory outbred mice ICR and rabbits”.

3. They use control groups in both mice and rabbits but the results obtained in them are not mentioned.

Answer:

Two control groups from 10 mice or 1 rabbit received normal PBS buffer as described in the revised section “Materials and methods”.  The results of fluorescent microscopy of brain samples of intact mice are shown on the revised figure 4. Autofluorescence of aromatic amino acid residues (phenyl alanine, tyrosine and tryptophane), biopolymers, living cells and organs at fluorescence excitation wavelength 550 nm and emission at 580 nm (that are typical for the fluorescent rhodamine dye RhoB used  to label BSA NP) is minimal (if any). 

 

4. In the manuscript the authors say that the nanoparticles are stored in different tissues and compare day 1 and day 2. In the conclusion of this study it is said “they accumulated mainly in brain and intestine with peak values in 1-2 days and subsequentdecline” lines 424-425.The results do not support this conclusion as there are no data beyond day 2.

Answer:

            The statement about “subsequent decline” in 2 days posttreatment has been removed from the revised “Conclusion” because of recommendation of other reviewer.  Line numbers were changed due to multiple corrections.

5. The text says “Available data on stability of protein NP in vitro permit to suggest their implementation in nanomedicine as diagnostic tools and potential drugs.”lines 335-336 Reference is needed.

Answer:

Our data on stability of protein NP in vitro were previously published in [9, 18] and are shown on the revised figures 1 and 2. Their implementation as diagnostic tools and potential drugs was suggested but not published yet. However, our suggestion is supported by the published data of other researchers and commercially available albumin-based nanomedicines approved by FDA (USA) as described in the revised text.

6. In the "fluorescent microscopy" methodology, it is necessary to specify the size of the sections obtained.

Answer:

Histological sections and prints had various irregular shapes and different sizes at different directions. Magnifications differed (600 for the former figure 1 and 400 for the former figures 4 and 5). The magnification of the fluorescent microscope was replaced to the scale bars on the revised figures 1, 4 and 5 according to the query of other reviewer.

7. Figures 1, 4 and 5 need a scale bar on the images.

Answer:

Scale bars were added into each image of the revised figures 1, 4 and 5.

 

8. 36% plagiarism has been detected, attached document, must be corrected

Answer:

Corrected.

 

 

Author Response File: Author Response.doc

Round 2

Reviewer 1 Report

The manuscript has been improved to a limited extent compared to Referees suggestions. In particular, the understanding of the protein shell effect on the biointeraction remained limited. In the answer to Referees comments, the Authors indicated that inorganic biodegradable nanoparticles do not have common features with their study, however this is not true because it is intrinsic of biodegradation that a protein corona forms on the nanoparticles (organic, inorganic or of biological origin it does not change this point). The protein corona will influence the biodistribution as well as the admnistration ratio, hence the comparison of biodistribution of biodegradable nanoparticles and of protein nanoparticles is very similar. Besides, there are a series of investigation techniques which allow monitoring inorganic nanoparticles more easily and accurately than fluorescent protein nanoparticles, because of leaching of fluorescent molecules. All these points, which were suggested to be implemented, have not been catched and discussed in the revised manuscript, which is not yet scientifically sound for publication. 

fine

Author Response

The manuscript has been improved to a limited extent compared to Referees suggestions. In particular, the understanding of the protein shell effect on the biointeraction remained limited. In the answer to Referees comments, the Authors indicated that inorganic biodegradable nanoparticles do not have common features with their study, however this is not true because it is intrinsic of biodegradation that a protein corona forms on the nanoparticles (organic, inorganic or of biological origin it does not change this point). The protein corona will influence the biodistribution as well as the admnistration ratio, hence the comparison of biodistribution of biodegradable nanoparticles and of protein nanoparticles is very similar. Besides, there are a series of investigation techniques which allow monitoring inorganic nanoparticles more easily and accurately than fluorescent protein nanoparticles, because of leaching of fluorescent molecules. All these points, which were suggested to be implemented, have not been catched and discussed in the revised manuscript, which is not yet scientifically sound for publication. 

 

Answer: 

  Thank you for your careful attention and valuable suggestions for revision of our manuscript. All your queries were taken into account and carefully addressed in the former revision. The only exception was about addition of 5 recommended references. New insertions in our re-revised manuscript are highlighted as Microsoft word track changes and additionally marked in green color for your convenience.

Our research was aimed at biodistribution of albumin NP among organs of laboratory mice and rabbits but not at protein shells on surface of other nanomaterials. Protein conformations are known to change on the solid supports including nanostructures.

We completely agree with the statement that “the protein corona will influence the biodistribution”. One could see the whole paragraph in the revised “Introduction”.

Bio-reactivity, cellular uptake and distribution among organs of  natural, synthetic and environmental  nanomaterials including both organic and inorganic NP  depend on surface proteins due to biocorona formation immediately after addition of biorelevant media or administration into organisms. Protein conformations can be changed on surfaces of nanostructures. Besides that coating with foreign proteins may induce innate and adaptive immune response [9]. Surface polyethylene glycol layer does not allow intracellular delivery of nanomaterials resulting in  their circulation in blood [11]. Delivery of nanometerials to brain tumours is mediated by NP biocorona in the bloodstream; leaky blood vessels at the tumour site; enhanced penetration and retention (EPR) in transformed cells; inhibition of drug efflux in endothelial and cancer cells; and active targeting by  means of specific ligands to receptors at the blood–brain barrier [12].Thus, apolipoprotein E  directs NP to the brain [13,14].

Undoubtedly that “there are a series of investigation techniques which allow monitoring inorganic nanoparticles more easily and accurately than fluorescent protein nanoparticles, because of leaching of fluorescent molecules”. However, the methods of isolation and identification of metallic NP cannot be used to monitor both the biodistribution and stability of undetectable protein NP.

All suggested references were carefully checked. The first paper https://www.sciencedirect.com/science/article/pii/S0021979721003908 published in Journal of Colloid and Interface Science 596 (2021) 332–341 describes silver-iron nanoparticles coated with polyethylene glycol but not with a protein. The second paper https://pubmed.ncbi.nlm.nih.gov/31293725/ published in Chem. Sci., 2019, 10, 5435-5443 is about PEGylated rhenium nanoclusters. The third article https://www.pnas.org/doi/10.1073/pnas.1911734116  (PNAS 2019, 117 (1) 103-113) is devoted to 2-step lifecycle of gold nanoparticles captured by primary fibroblasts in vitro but not in vivo. The fourth article https://pubs.acs.org/doi/abs/10.1021/acsnano.5b00042 (ACS Nano 2015, 9, 8, 7925–7939) represents study of long-term fate of gold/iron oxide heterostructures covered with two different coating shells - amphiphilic polymer or polyethylene glycol but not with a protein after intravenous injection in mice.           The fifth one https://pubmed.ncbi.nlm.nih.gov/32877170/ published in ACS Nano 2020, 14, 10, 12840–12853 describes gold-iron alloys but not protein NP or other nanomaterials with protein coating.

            Both “Introduction” and “Discussion” were revised according to your recommendations.

 

Author Response File: Author Response.doc

Reviewer 2 Report

The auhors have made the changes suggested therfore, the manuscript can be accepted in the present form.

Author Response

We greatly appreciate your feedback and recommendations, which allowed us to revise our manuscript and to improve its content.

Author Response File: Author Response.pdf

Reviewer 3 Report

 

This paper has been significantly improved and the answers and clarification of the authors are convincing.

 

Still a little remark to avoid confusion among the readers.  In my review, I specified: “The term RhoB may be confusing for cell biologists and should probably be adapted to avoid this problem.” The authors do not answer.  If you type RhoB on your computer (google or another), you will see first appearing “RHOB is a member of the Rho GTP protein family”, but not Rhodamine B.  Please clarify.

 

In addition, my feeling is that several convincing answers to my criticisms could or should be included in the paper, since they provide strong arguments and/or explanations.

 

 

 

Author Response

This paper has been significantly improved and the answers and clarification of the authors are convincing.

Answer:

Thank you for your careful attention and valuable suggestions for revision of our manuscript. New insertions in our re-revised manuscript are highlighted as Microsoft word track changes and additionally marked in green color in order to distinguish the first and the second rounds of our corrections.

Still a little remark to avoid confusion among the readers.  In my review, I specified: “The term RhoB may be confusing for cell biologists and should probably be adapted to avoid this problem.” The authors do not answer.  If you type RhoB on your computer (google or another), you will see first appearing “RHOB is a member of the Rho GTP protein family”, but not Rhodamine B.  Please clarify.

Answer:

According to your advice the information about advantages of the fluorescent rhodamine dye  RhoB including its stability and fluorescence excitation/emission spectra that do not overlap with autofluorescence of proteins from living cells and organs were added in the section “Materials and methods” of the re-revised manuscript.

 

In addition, my feeling is that several convincing answers to my criticisms could or should be included in the paper, since they provide strong arguments and/or explanations.

Answer:

“Discussion” were revised according to your recommendations.

 

 

 

 

Author Response File: Author Response.doc

Reviewer 4 Report

The authors have made the required changes

Author Response

The authors have made the required changes

Answer:

We greatly appreciate your attention, research interest and valuable suggestions for improvement of our manuscript and even for further development.

 

Author Response File: Author Response.doc