In Vitro Drug Delivery through the Blood–Brain Barrier Using Cold Atmospheric Plasma

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript is an interdisciplinary study of physics and biology. Since I don't know much about plasma, I will mainly comment on the biology part.

The content is simple, the experimental part of this manuscript is very detailed, however, there are omissions in the experimental design, and the analysis and interpretation of some data are also lacking, so I’d recommend a major revision of the draft.

 

Major:

1.      In Fig. 4, why the voltage values are not matched for BBB cell and bEND.3 cells? In addition, it’s hard to say “a significant increase” in treated groups, especially for BBB cells. Unless the author compares with similar data from other literature and the difference of control and treated groups is more obvious than most other data.

2.      In Fig. 5, from (a) to (d), it seems that not only the fluorescence intensity increases, but also the cell morphology changes significantly. Can the authors explain this and cite appropriate references? In addition, please quantify the fluorescence intensity in the figure to compare the differences between different groups.

3.      Can the authors give more data to verify ROS might damage tight junction proteins, for example, western blot assay?

4.      Are the experiments triple? Especially the cell viability measurement? I believe not since the authors didn’t mention in the experimental part. For cell viability assay, 3-6 samples for each group are necessary.

 

Minor:

1.      The author often repeats abbreviations. Please delete the redundant abbr or full name. For example, line 57 and line289, “(RONS)”; line 167 and line 315, “(ROS)”; line 179 and line 194, “(PBS)”; line 283, “(BBB)”;

2.      Format problem: line 266-267; line 313, “Blood”;

3.      Line 232, what’s the meaning of “insert” and “well”?

4.      Please provide the DOI for all the reference as possible, for example, ref 19 and 43.

 

Author Response

1.    In Fig. 4, why the voltage values are not matched for BBB cell and bEND.3 cells?

In addition, it’s hard to say “a significant increase” in treated groups, especially for BBB cells. Unless the author compares with similar data from other literature and the difference of control and treated groups is more obvious than most other data.

 

RESPONSE: The two plasma devices; plasma jet and dielectric barrier discharge (DBD) plasma were used for BBB cell model and bend.3 cell culture respectively. Generally we use spiral wire microplasma for generating plasma components (e.g. RONS). As BBB was ready-made culture on 24-transwell plate and the culture area of each insert was very small, we had to use plasma device with argon (Ar) gas flow ( it was difficult to use DBD plasma in small area of culture insert). To generate plasma in Plasma Jet device with Argon gas, we used 10kHz frequency and 2 -3kV0-p discharge voltage for making drug permeability. (Among our trials this conditions performed better result in drug permeability). In plasma Jet higher frequency is needed to generate plasma compared to DBD plasma. The more frequency is used, the less voltage is needed to generate plasma. On the other hand, DBD plasma was generated with 5kHz frequency and 2-4 kVp-p discharge voltage. In short, due the the structure difference of plasma Jet and DBD plasma, the voltage value and frequency value were different.

A calibration curve has been made to determine the concentration of drug passed through the bEND.3 cell layer. It was done for bEND.3 only, not BBB. As BBB was primary cells and ready-made co-culture cells, We do not have any stock. However, drug concentration in plasma treated cells was higher than that of control cells (figure 04b). Line 248-251.

 

2.    In Fig. 5, from (a) to (d), it seems that not only the fluorescence intensity increases, but also the cell morphology changes significantly. Can the authors explain this and cite appropriate references? In addition, please quantify the fluorescence intensity in the figure to compare the differences between different groups.

RESPONSE: For quantitative value of florescence intensity of ROS, the image of whole plate was taken in microscope and converted to quantitative value using imageJ software. Line 192.

 

3.      Can the authors give more data to verify ROS might damage tight junction proteins, for example, western blot assay?
RESPONSE: This study only focused on drug permeability. However, several research group confirmed that ROS causes damage to tight junction proteins. Line 274-276, 331-336.

 

4.      Are the experiments triple? Especially the cell viability measurement? I believe not since the authors didn’t mention in the experimental part. For cell viability assay, 3-6 samples for each group are necessary.

RESPONSE: Yes, all the experiments were done at 3 different times with triplicate (3) sample each time for each group. All those figure was shown with error bar. It means data are triplicate. However, the statement “Triplicate experiments for each sample were conducted for ensuring reproducibility” has been included in the method sections shown in last line of 2.5, 2.6, 2.7 and 2.8 of method section.

 
Minor:
1.    The author often repeats abbreviations. Please delete the redundant abbr or full name. For example, line 57 and line289, “(RONS)”; line 167 and line 315, “(ROS)”; line 179 and line 194, “(PBS)”; line 283, “(BBB)”;

RESPONSE: Abbreviation problem is solved.

2.    Format problem: line 266-267; line 313, “Blood”;

RESPONSE: Format problem solved. Now line is 301.

3.    Line 232, what’s the meaning of “insert” and “well”?

RESPONSE: Trans-well plate contains two chambers; apical and basolateral. The apical chamber is called insert. This insert contain membrane that support cell culture and growth. The membrane of the insert has pore spacing that allows the permeation of drugs through a compromised cell membrane layer to the bottom chamber called the basolateral chamber or well. Line is now 255.

4.    Please provide the DOI for all the reference as possible, for example, ref 19 and 43.

RESPONSE: We did. Although two of them lack DOI. Instead PMID was provided.

 

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

No significant differences were analyzed for the differences between all treatments.

Figure 5 can add bright field photos to determine the number of cells.

Figure 6 only analyzes the cell survival rate after 12 hours of treatment, which is obviously not enough to draw a conclusion.

Author Response

1.    No significant differences were analyzed for the differences between all treatments.

RESPONSE: A calibration curve has been made to determine the concentration of drug passed through the bEND.3 cell layer. It was done for bEND.3 only, not BBB. As BBB was primary cells and ready-made co-culture cells, We do not have any stock. However, drug concentration in plasma treated cells was higher than that of control cells (figure 04b).

2.    Figure 5 can add bright field photos to determine the number of cells.

RESPONSE: Bright field photos has been added in figure 5. The bar graph of florescence intensity of ROS has been added in figure 5. For quantitative value of florescence intensity of ROS, the image of whole plate was taken in microscope and converted to quantitative value using imageJ software. Line 192.

3.    Figure 6 only analyzes the cell survival rate after 12 hours of treatment, which is obviously not enough to draw a conclusion.

RESPONSE: Cell survival rate is now shown after 24 hours of plasma irradiation. 24 hours is enough to multiply the cells and thereby replace the dead cells. After 24 hours cell viability of plasma treated cells was similar to control group.

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

This study explored the potential of cold atmospheric plasma (CAP) to enhance drug delivery to the brain by potentially disrupting the blood-brain barrier (BBB) through the generation of reactive oxygen and nitrogen species (RONS). The experiment utilized an in vitro BBB model and an immortalized cell line to test drug permeability, with results showing decreased trans-endothelial electrical resistance (TEER) after CAP treatment. The study highlighted the potential of CAP as a method to improve brain drug delivery for neurological disorders. However, there are still a few issues before the authors can make their claim.

What are the problems for FUS, nanoparticles, and peptides for the delivery passing BBB? The authors should give a more detailed review in the introduction.

What would the strategy be for in vivo experiments to apply CAP for BBB disruption?

Measuring cell viability with trypan blue is not an accurate method. I suggest the authors perform other measurements such as MTT assay.

Since the DCFH-DA assay is a quantitative method, the degree of oxidative stress under each condition should be noted (for example, a bar graph representing the FL intensities).

In Figure 4, it seems that the fluorescence intensity after CAP treatment merely increased by 30 to 60%. This can hardly support the claim for a higher drug passage considering the background in the plate reader. The authors should also make a calibration curve to determine the concentration of FD-4 in each cell sample.

 

Comments on the Quality of English Language

There are quite a few grammar issues in the manuscript. I suggest the authors carefully check these. 

Author Response

1. What are the problems for FUS, nanoparticles, and peptides for the delivery passing BBB? The authors should give a more detailed review in the introduction.

RESPONSE: Thank you very much for your nice question. The drawback of those three methods has been discussed in introduction in line number 40-43, 45-49, and 51-54. 

2. What would the strategy be for in vivo experiments to apply CAP for BBB disruption?

RESPONSE: It has been discussed in conclusion part. “Moreover, in vivo study can also be done in animal blood vessel by using indirect plasma treatment by intravenous injection of plasma treated components”. Line number is 364-367.

3. Measuring cell viability with trypan blue is not an accurate method. I suggest the authors perform other measurements such as MTT assay.

RESPONSE: Yes, Trypan blue is toxic to the cells when the cells is incubated for more than 10 minutes Kim et al. (2016). In our study, we counted the cells immediately after adding the trypan blue in the cells. Line number is 216. As reported by Calero et al. (2014) the data for MNP cytotoxicities using the trypan blue assay are consistent with the data obtained for the MTT assay, which is often considered as the gold standard.

4. Since the DCFH-DA assay is a quantitative method, the degree of oxidative stress under each condition should be noted (for example, a bar graph representing the FL intensities).

RESPONSE: The bar graph of florescence intensity of ROS has been added in figure 5. For quantitative value of florescence intensity of ROS, the image of whole plate was taken in microscope and converted to quantitative value using imageJ software. Line 193.

5. In Figure 4, it seems that the fluorescence intensity after CAP treatment merely increased by 30 to 60%. This can hardly support the claim for a higher drug passage considering the background in the plate reader. The authors should also make a calibration curve to determine the concentration of FD-4 in each cell sample.

RESPONSE: A calibration curve has been made to determine the concentration of drug passed through the bEND.3 cell layer. It was done for bEND.3 only, not BBB. As BBB was primary cells and ready-made co-culture cells, We do not have any stock. However, drug concentration in plasma treated cells was higher than that of control cells (figure 04b).

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors solved all my concerns.

Author Response

Dear Reviewer,

Thank you very much for your positive comment.

Reviewer 2 Report

Comments and Suggestions for Authors

The author responded positively to my suggestions. However, the differences between each treatment group and the control group have not yet been tested for significance. This statistical analysis is necessary and needs to be supplemented by the authors.

Author Response

Dear Reviewer,

Thank you very much for your positive comment. The differences between each treatment group and the control group were tested for significance using the student T-test and added in Figures 3, 4, 5, and 6.

Reviewer 3 Report

Comments and Suggestions for Authors

I see the authors' effort in addressing the comments and improving the quality of this paper. However, my previous comments 3 and 5 remain unresolved. 

In Figure 4B, a calibration curve was added. What does this curve tell us? What is the detection limit? How accurate would it be? Can you illustrate the reproducibility? 

For this work to be published, I think more experiments must be performed. 

 

Author Response

RESPONSE:

Dear Reviewer

Thank you very much for your comments. The revision was done according to the questions.

“I see the authors' effort in addressing the comments and improving the quality of this paper. However, my previous comments 3 and 5 remain unresolved. 
In Figure 4B, a calibration curve was added. What does this curve tell us? What is the detection limit? How accurate would it be? Can you illustrate the reproducibility?"

Comment 3: Measuring cell viability with trypan blue is not an accurate method. I suggest the authors perform other measurements such as MTT assay.

RESPONSE: In addition to measuring cell viability with trypan blue, cell death was also measured with propidium iodide using flow cytometer. The experimental methods and results have been discussed in 2.8 and 3.4 section (figure 07) respectively (blue color marked).
Comment 5: In Figure 4B, a calibration curve was added. What does this curve tell us? What is the detection limit? How accurate would it be? Can you illustrate the reproducibility? 
RESPONSE: 
(a) In Figure 4B (in manuscript), the line graph indicates the concentration of drug passed to the basolateral part of trans-well plate. The concentration was calculated using calibration curve. The concentration of permeated drug was higher in plasma treated samples compared to control group.
(b) The lowest concentration of the analyte that can be reliably detected (detection limit, LOD) was 0.184. Where 
Plasma Discharge         Concentration 4µg/ml
Control        0.1239
2.0 kv        0.35
3.0 kV        0.9607
4.0 kV        1.214

(c) As the figure added below in this response, the accuracy is 0.9997 which is closer to 1. Coefficient of Determination (R²) ranges from 0 to 1, where a value closer to 1 indicates a better accuracy. (Figure a)

(d) Triplicate experiments for each sample were conducted for ensuring reproducibility. Error bar was added to refer the triplicate experiment (Figure b)

 
Figure: Calibration curve (a) and line graph for drug concentration (b).

 

 

Author Response File: Author Response.docx

Round 3

Reviewer 3 Report

Comments and Suggestions for Authors

I thank the authors' efforts in addressing my previous round of comments. The manuscript looks good now and can be published as is.