Synthesis and Application of Albumin Nanoparticles Loaded with Prussian Blue Nanozymes

Round 1

Reviewer 1 Report

The idea of synthesizing albumin nanoparticle for enhancing stability of Prussian blue nanozymes is quite insightful yet the manuscript is not acceptable in current form and requires major changes to be done before consideration to publication.

Comments

1. In abstract section, author mentioned “at high ethanol concentration solubility of ethanol decreases”, the reviewer could not get the author’s point. The reviewer suggests reconsidering the whole abstract and representing it in more informative way.
2. In section 2.3, preparation of nanozyme loaded BSA nanoparticles, author mentioned that BSA containing solution was heated at 70°C, which may denature the BSA protein and thus making preparation protocol more suspicious. The reviewer suggests reconsidering the protocol. Also, in preparation part, reviewer could not get the meaning of PB nanoparticle, as its abbreviation is not present before anywhere in the manuscript.
3. In section 2.4, what is the point of determining concentration of nanoparticles by gravimetric analysis, if it is not discussed in the result section?
4. In result section, there is need of incorporation of DLS actual graphs for more authentications of results (at least of finalized nanoparticles in main manuscript and all others in supplementary information). Moreover, Author mentioned that “2 g of Prussian blue nanozymes with mean hydrodynamic diameter of 44 nm and PDI of 0.167”, but there is no relevant data in the form of DLS graph or table is present in anywhere of manuscript.
5. The reviewer does not able to understand the relationship between concentration and size of BSA nanoparticles, as author mentioned that with increase in concentration of BSA, the size of resultant nanoparticle will also increases. Please provide the meaningful explanation for this statement.

Author Response

Dear Editor-in-Chief and Referees

We express our great gratitude to the Reviewers for comments and thoughtful suggestions. Based on these comments and suggestions, we have made modifications to the original manuscript.

Key changes we made are summarized below:

1. Abstract was re-written: more details have been added
2. Size distribution plots were added (Fig. 1G, Fig. S2, S4, S7)

All changes are highlighted with yellow.

We believe that the manuscript has been improved and hope it has reached high journal's standard.

Sincerely yours

Pavel Khamtsov.

Reviewer #1

The idea of synthesizing albumin nanoparticle for enhancing stability of Prussian blue nanozymes is quite insightful yet the manuscript is not acceptable in current form and requires major changes to be done before consideration to publication.

Comments

1. In abstract section, author mentioned “at high ethanol concentration solubility of ethanol decreases”, the reviewer could not get the author’s point. The reviewer suggests reconsidering the whole abstract and representing it in more informative way.

Response:

The correct sentence is “…at high ethanol concentration solubility of albumin decreases…”. We changed this sentence and re-wrote abstract in accordance with Reviewer’s suggestion.

2. In section 2.3, preparation of nanozyme loaded BSA nanoparticles, author mentioned that BSA containing solution was heated at 70°C, which may denature the BSA protein and thus making preparation protocol more suspicious. The reviewer suggests reconsidering the protocol.

Response:

The protocol of albumin nanoparticle preparation is based on a well optimized method proposed by Weber and colleagues [Weber, C., Coester, C., Kreuter, J., & Langer, K. (2000). International journal of pharmaceutics, 194(1), 91–102. https://doi.org/10.1016/s0378-5173(99)00370-1]. The authors demonstrated that albumin nanoparticles prepared by ethanol desolvation can be stabilized not only using cross-linker (e.g. glutaraldehyde or EDC) but also with the aid of heating for 2 h at +70 C. We used heat-mediated nanoparticle stabilization because addition of glutaraldehyde in the course of ethanol desolvation resulted in severe aggregation. Heating of hydroethanolic albumin solution leads to formation inter-molecule disulfide bonds, which are responsible for stabilization of nanoparticles (Khramtsov, P., Kalashnikova, T., Bochkova, M., Kropaneva, M., Timganova, V., Zamorina, S., & Rayev, M. (2021). International journal of pharmaceutics, 599, 120422. DOI 10.1016/j.ijpharm.2021.120422). Indeed, denaturation of albumin occurs upon heating, but it is not interfere with nanoparticles stability or applicability in immunoassays. Moreover, heat-stabilized albumin nanoparticles were successfully applied for drug delivery purposes (for example, Yu et al. Nanoscale Research Letters 2014, 9:343).

Also, in preparation part, reviewer could not get the meaning of PB nanoparticle, as its abbreviation is not present before anywhere in the manuscript.

Response:

Fixed (Section 2.3.)

3. In section 2.4, what is the point of determining concentration of nanoparticles by gravimetric analysis, if it is not discussed in the result section?

Response:

Determination of nanoparticles is necessary for their functionalization and between-batches comparison. We described the method for nanoparticle concentration measurement because some commonly used methods (e.g. Bradford protein assay) can yield erroneous results (discussed in Khramtsov, P., Kalashnikova, T., Bochkova, M., Kropaneva, M., Timganova, V., Zamorina, S., & Rayev, M. (2021). International journal of pharmaceutics, 599, 120422. DOI 10.1016/j.ijpharm.2021.120422). Besides, in some papers the method of nanoparticle quantification is not specified, which makes these papers hardly reproducible. In addition, concentration of nanoparticles needs to be measured to determine the yield, loading capacity, and encapsulation efficiency (Table 1)

4. In result section, there is need of incorporation of DLS actual graphs for more authentications of results (at least of finalized nanoparticles in main manuscript and all others in supplementary information). Moreover, Author mentioned that “2 g of Prussian blue nanozymes with mean hydrodynamic diameter of 44 nm and PDI of 0.167”, but there is no relevant data in the form of DLS graph or table is present in anywhere of manuscript.

Response:

Representative intensity-weighted size distribution (measured by dynamic light scattering) have beed added to the manuscript and Supplementary materials

5. The reviewer does not able to understand the relationship between concentration and size of BSA nanoparticles, as author mentioned that with increase in concentration of BSA, the size of resultant nanoparticle will also increases. Please provide the meaningful explanation for this statement.

Response:

Relationship between starting albumin concentration and resulting nanoparticle size was studied and explained in paper by F. Galisteo-González and J. A. Molina-Bolívar (Colloids Surf B Biointerfaces . 2014 Nov 1;123:286-92. doi: 10.1016/j.colsurfb.2014.09.028, Section 3.2.). Concentrated BSA solutions have higher viscosity, which slows down transport of BSA molecules between water and ethanol, which in turn leads to slower nucleation rate. The lower the number of nuclei the larger the resulting nanoparticles. At very high BSA concentration, despite large number of nuclei present, their growth is not limited because BSA presents in excess. Besides, the chance of inter-particle collisions and formation of large aggregates is higher.

We performed synthesis of nanoparticles using three starting BSA concentrations (20,30, and 40 mg/ml) in the presense of Prussian blue nanozymes. The same dependence was observed: the higher BSA concentration the large nanoparticles. Reference to the mentioned paper was added to the manuscript.

Reviewer 2 Report

This paper reports a method to encapsulate Prussian Blue within albumin nanoparticles. It is interesting that this protein encapsulated Prussian blue can be further conjugated with antibodies to be used in ELISA. But it is not clear whether this nanozyme improve the detection sensitivity as compared to HRP or other types of nanozymes.

1. Abstract: “At a high ethanol concentration solubility of ethanol decreases resulting in the formation of albumin nanoparticles loaded with nanozymes.” Please explain what the meaning of “at high ethanol concentration, the solubility of ethanol decreases”.
2. Please explain clearly the novelty and advantages of using albumin nanoparticles to encapsulate PB nanozymes.
3. Figure 1: What does empty nanoparticles (PB@BSA0) referring to? How is this different from PB 38nm? This label is very confusing. Maybe it is clearer to just call it BSA capsule.
4. Figure 1: pls label the tubes in E.
5. Figure 4: the caption claims SD of 3 technical replicates are presented. But no error bars were found the calibration plots.
6. Figure 5d should be replotted with time as the x-axis to make clear that we are looking at the optimal incubation time here.
7. Figure 6: the authors plotted three calibrations for PB@BSA5, there is not average and SD involved, pls remove this statement from the caption “mean±SD”.
8. The authors should comment on the Limit of Detection achieved for such PSA sensor. Is it sufficient for clinical applications?

Author Response

Dear Editor-in-Chief and Referees

We express our great gratitude to the Reviewers for comments and thoughtful suggestions. Based on these comments and suggestions, we have made modifications to the original manuscript.

Key changes we made are summarized below:

1. Abstract was re-written: more details have been added
2. Size distribution plots were added (Fig. 1G, Fig. S2, S4, S7)

All changes are highlighted with yellow.

We believe that the manuscript has been improved and hope it has reached high journal's standard.

Sincerely yours

Pavel Khamtsov.

Reviewer #2

This paper reports a method to encapsulate Prussian Blue within albumin nanoparticles. It is interesting that this protein encapsulated Prussian blue can be further conjugated with antibodies to be used in ELISA. But it is not clear whether this nanozyme improve the detection sensitivity as compared to HRP or other types of nanozymes.

1. Abstract: “At a high ethanol concentration solubility of ethanol decreases resulting in the formation of albumin nanoparticles loaded with nanozymes.” Please explain what the meaning of “at high ethanol concentration, the solubility of ethanol decreases”.

Response:

The correct sentence is “at high ethanol concentration solubility of albumin decreases”. We changed this sentence and re-wrote abstract.

2. Please explain clearly the novelty and advantages of using albumin nanoparticles to encapsulate PB nanozymes.

Response:

Novelty and advantages of proposed method are described in the Introduction section, paragraphs 3 and 4. More specifically, this approach is not completely novel, because desolvation technique has been previously applied to encapsulate nanoparticles. However, this method was not used to encapsulate prussian blue nanozymes. Prussian blue possess some specific properties, first of all, instability at neutral and alkaline pH. At the same time, desolvation is usually performed in slightly alkaline conditions (pH 8-9). Alkaline pH provides negative charge of BSA molecules, which decreases the chance of uncontrolled albumin aggregation upon ethanol addition. These synthesis conditions are incompatible with prussian blue, therefore their optimization was necessary. We performed desolvation of albumin in deionized water without pH adjustment. In these conditions, hydrolysis of prussian blue was not detected.

Advantages of desolvation method are simplicity, scalability, and good reproducibility (Langer, K et al. International journal of pharmaceutics vol. 347,1-2 (2008): 109-17. doi:10.1016/j.ijpharm.2007.06.028). Albumin nanoparticles synthesized by the desolvation method have good stability in buffers, which are used in the course of functionalization and immunoassays. Besides, albumin molecules contain multiple functional groups such as amine, thiol, carboxyl, which can be used for the covalent attachment of recognition molecules.

Novelty of the present work has been added in the Introduction section (paragraph 3).

Figure 1: What does empty nanoparticles (PB@BSA0) referring to? How is this different from PB 38nm? This label is very confusing. Maybe it is clearer to just call it BSA capsule.

Response:

Label of BSA nanoparticles, which do not contain nanozymes, was changed from PB@BSA0 to “BSA NP”

Figure 1: pls label the tubes in E.

Response:

Tubes were labelled.

Figure 4: the caption claims SD of 3 technical replicates are presented. But no error bars were found the calibration plots.

Response:

In this analysis error bars are too small and hidden by the data points. We added explanation in the figure capture.

Figure 5d should be replotted with time as the x-axis to make clear that we are looking at the optimal incubation time here.

Response:

We tried to plot Absorbance vs time, but plot was still not clear enough. Therefore, we changed it in another way. Calibration curves obtained in 5 min time intervals are shown. Measurements that were performed after 30 min were removed. Now, it can be seen that signal reaches maximum in 25-30 min. Full version can be found in Supplementary information (Figure S9)

Figure 6: the authors plotted three calibrations for PB@BSA5, there is not average and SD involved, pls remove this statement from the caption “mean±SD”.

Response:

Error bars are too small and hidden by the data points. We added explanation in the figure capture. Additionally, reproducibility can be assessed from Figure S12, which contains data points for each technical replicate.

The authors should comment on the Limit of Detection achieved for such PSA sensor. Is it sufficient for clinical applications?

Response:

Clinically relevant threshold for PSA is 4 ng/ml (Thompson, Ian M et al. The New England journal of medicine vol. 350,22 (2004): 2239-46. doi:10.1056/NEJMoa031918). Determination of PSA up to several tens of ng/ml is also essential for the diagnostic purposes (Santok, Glen Denmer R et al. “Prostate-specific antigen 10-20 ng/mL: A predictor of degree of upgrading to ≥8 among patients with biopsy Gleason score 6.” Investigative and clinical urology vol. 58,2 (2017): 90-97. doi:10.4111/icu.2017.58.2.90). Developed assay allows detection of PSA at mentioned concentrations. Nevertheless, we do not claim clinical applicability of the assay because it requires much more strict validation and optimization. Commercial test kits (mostly ELISAs) allow PSA detection from about 0.1 ng/mL. The goal of this work was not to develop more sensitive immunoassay, but rather to demonstrate principal applicability of nanoparticles in colorimetric assay.

 We added comments on applicability of the PSA assay in the end of the Results and Discussion section.

Reviewer 3 Report

The work is interesting, very clearly presented. It can be accepted for publication.

Minor remarks:

1) Abstract, lines 15-1: "At a high ethanol concentration solubility of ethanol decreases..". Is it correct? Do you mean "..solubility of albumin decreases..", possibly?

2) Section 2.5.: Is the definition of Yield correct? I would expect the inverse, 

3) Section 2.7, line 163: The term A450 should be defined and described haw it was measured (instrumentation).

Author Response

Dear Editor-in-Chief and Referees

We express our great gratitude to the Reviewers for comments and thoughtful suggestions. Based on these comments and suggestions, we have made modifications to the original manuscript.

Key changes we made are summarized below:

1. Abstract was re-written: more details have been added
2. Size distribution plots were added (Fig. 1G, Fig. S2, S4, S7)

All changes are highlighted with yellow.

We believe that the manuscript has been improved and hope it has reached high journal's standard.

Sincerely yours

Pavel Khamtsov.

Reviewer #3

The work is interesting, very clearly presented. It can be accepted for publication.

Minor remarks:

1) Abstract, lines 15-1: "At a high ethanol concentration solubility of ethanol decreases..". Is it correct? Do you mean "..solubility of albumin decreases..", possibly?

Response:

The correct sentence is “at high ethanol concentration solubility of albumin decreases”. We changed this sentence and re-wrote abstract.

2) Section 2.5.: Is the definition of Yield correct? I would expect the inverse,

Response:

The equation was incorrect. The numerator and denominator were reversed. We fixed the equation.

3) Section 2.7, line 163: The term A450 should be defined and described haw it was measured (instrumentation).

Response:

A450 stands for value of absorbance at 450 nm. It is measured using spectrophotometer. Definition was added (Section 2.7), instrumentation is listed in the Section 2.1.

Round 2

Reviewer 1 Report

The authors have responded to the comments and made changes in the revised manuscript. The manuscript should be accepted in present form.

Reviewer 2 Report

The revision is ok.