Investigation of Plasmid DNA Delivery and Cell Viability Dynamics for Optimal Cell Electrotransfection In Vitro

Round 1

Reviewer 1 Report

No more comments

Reviewer 2 Report

The authors performed the main recommendations. 

Reviewer 3 Report

The authors have fully addressed my concerns.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

In this manuscript, the authors worked on the optimization of electroporation in mammalian cells. Within the Chinese Hamster Ovary cell line CHO-K1, the authors investigated the role of plasmid concentration in transfection efficiency and cell viability and concluded that the plasmid concentration is positively correlated with transfection efficiency and negatively correlated with cell viability. They also tried to determine the plasmid concentration as the distance between electroporated cell membrane and the nearest plasmid and proposed that the distance between cell membrane and the nearest plasmid is a more accurate measurement to predict the electroporation efficiency. Some ideas are interesting. However, the whole study is lack of novelty and seems not fully supported by the evidence. This manuscript can be improved in the following aspects.

Major concerns

A major conclusion of this manuscript is the inverse dependence of electrotransfection efficiency and cell viability on plasmid concentration. However, it is already well-known. Furthermore, because of the cell type specificity, the optimized plasmid concentration for CHO-K1 cell electroporation with the certain parameters optimized in this study may not suit for other cell types. The idea of measuring the distance between the cell membrane and the nearest plasmid to predict electroporation efficiency is interesting. However, there is not enough evidence in this manuscript to support this idea. The authors need to distinct the changes of plasmid concentration and that of the nearest distance between cell membrane and plasmid. An easy way to achieve this is adjusting the cell number while maintain the plasmid concentration to have different nearest distance between cell membrane and plasmid. If the electroporation efficiency and cell viability have the same correlation to the nearest distance between cell membrane and plasmid, by adjusting the plasmid concentration or the cell number, it could be a good evidence to support the hypothesis. Following the point above, the calculation of total cell volume is not accurate. The diameter was measured in cells attached on coverslips, which is much flatter than floating cells. So the calculated cell volume can be much bigger than their actual size. Measuring the individual cell size by flow cytometry or simply measuring the volume of cell pallet can be better options.

Reviewer 2 Report

In this manuscript, entitled- “Investigation of plasmid DNA delivery and cell viability dynamics for optimal cell electro-transfection in vitro”, by Chopra et al. demonstrates electro-transfections of plasmid DNA into cells. The purpose of this study is to determine the correlation-based dependency on the distance between cell membrane and DNA molecule with the transfection efficiency and cell viability. The author evaluates the transfection efficiency and cell viability by changing concentration of plasmid DNA and high voltage electric pulses. The study revealed that the transfection efficiency increases with the increase in concentration of plasmid. Also noted that electric pulses greatly influence the transfection efficiency, higher the electric pulse higher is the transfection efficiency. The study suggests that cell viability decreases with the increase in concentration of plasmid DNA; a similar trend was observed i.e. cell viability decreases with the increase in electric pulse. However, at the final section of the study (Fig 4 & 5) they propose that the distance between the cell membrane and the DNA molecule would be an accurate measurement in order to understand transfection efficiency. The study nicely demonstrates the aforementioned factors on electro-transfection of big molecule like plasmid DNA. The results of this study would are valuable information. The manuscript is written very well and has done systematic study on this particular issue.

Minor comments:

The author didn’t describe clearly the differences in experiments describe in Figure 1 (transfected cell that do not account for cell viability) and 3 (all treated cell). If it is possible please show an image of cell and how the ‘r’ was calculated, it would be clear for a broader range of audiences.

 

Reviewer 3 Report

The manuscript applsci-696061 entitled “Investigation of plasmid DNA delivery and cell viability dynamics for optimal cell electrotransfection in vitro” investigates the combined study in which the dynamics of both evaluation types of transfection efficiency and the cell viability were evaluated in dependence of plasmid concentration as well as at the different number of high voltage (HV) electric pulses. In addition, the authors verified a dependence of the transfection efficiency and cell viability on the distance between the cell membrane and the nearest plasmid. In my opinion, this manuscript needs some revision before to be considered for publication.

Major changes:

The authors claimed in line 58 “no consistent research has been published on the dependence of electrotransfection efficiency on plasmid concentration.” But the next sentence is contradictory “Nevertheless, there are a few publications that show an improvement in the electrotransfection efficiency with the increase in plasmid concentration.” Also line 62 and 63 “Others report the increase in cell electrotransfection efficiency with higher plasmid concentrations and note that the cell viability decreases to ~ 70–80 %...” These statements should be completed/improved, in order to clarify and highlight the difference between the present study and the previous ones, and the authors must make clear what is the novelty of the present study.

 

The authors claimed in line 71 of the Introduction “For the first time, we show a quantitative, correlation-based dependency on the distance between the cell and plasmid DNA molecule with cell transfection efficiency and with cell viability.” However, it is mandatory to clarify the dependency of this correlation regarding to the cell radius and plasmid size/molecular weight.

 

This work seems too much preliminary and need some experiments to complement some important information. As it is well described in line 37 of Introduction “…electroporation of the cell membrane can be reversible or irreversible.” Some controls should be shown regarding to the cell viability, without plasmid presence, under different electric pulsed used (1, 2 and 3 HV). Only comparing this result with the other experiments it can be mentioned that the effect observed in cell viability could be related to the pDNA amount and not with the electric pulses used…

 

Also a control should be included about the influence of electric pulses into the pDNA stability, for instance, showing in an electrophoresis agarose gel the pDNA samples at different concentrations, before and after be submitted to the different electric pulses. This result can be useful to understand if lower influence in the transfection efficiency observed in some experiments can be related or not with some plasmid degradation under the experimental conditions. This kind of information it will be important to take in consideration to define the ideal conditions of pDNA concentration and electric pulses to be used regarding some kind of therapeutic applications.

 

It should be indicate in Discussion or Conclusion sections, for instance, the recommended optimal conditions to apply electrotransfection to obtain satisfactory transfection efficiency but avoiding decrease of cell viability, or pDNA degradation.

 

In addition, some experiments (related to apoptosis) should be performed for the optimal conditions for example to prove that these conditions did not activate/induce programmed cell death. Unless the aim be the elimination of cancer cells, in other studies it is not the intended therapeutic effect.

 

A comparison study with other transfection procedure such as the calcium chloride and thermal shock should be valuable to evaluate and highlight the transfection efficiency of electroporation method.

Minor changes:

Some corrections should be included throughout the manuscript, specially in methods section, for instance:

It is missing a punctuation in the end of line 68. Correct the chemical formula of CO2 in line77, the same in line 91. The cell number is not 104 (line 103 and 110). Correct the temperature unit of line 108.

Reviewer 4 Report

The paper presents the gene trasfection by means of high voltage pulses. The topic is interesting since authors use classical ECT pulses in gene transfection.

Minor remarks

pp. 3 line 103 and 110 Please check cell concentrtion x104...

line 103: what is the plasmid concentration in the 5uL

pp.4 line 155 what is the meaning of 25 at th end of the line

line 158 what is the meaning of the acronym 'HV'

Major remarks:

Why the authors didn't discuss the classical gene trasfection obtained using the low voltage long time pulses?

What difference could be remarque with the presented electroporation procedure? What is the difference with classical procedure?

Please, consider also the paper in the introduction

Gehl, “Electroporation: theory and methods, perspectives for drug delivery, gene therapy and research,” Acta Physiologica Scandinavica, vol. 177, no. 4, pp. 437–447, Apr. 2003

Why plasmide concentration affect cell viability?

Considering cell viability could the authors evaluate the real trasfections? Why there is a higher transfection and a lower viability? please, discuss this topic.