Review Reports

Round 1

Reviewer 1 Report

Authors present a created electronic emulation system based on ex vivo results that are published on another paper (Langus et. Al. 2016). I do agree that it is indeed an issue of great interest to calibrate the electric impedance and resistance at different treatments (tissues, electrode surface contact and ect.), since the effect of generated electric fields on cells in tissues will be different. This way the level of electroporation will differ.

 

However, there are some point in this paper that needs to be addressed:

Firstly, the title of the article is not accurate when mentioning “biological load”. If I understand correctly it is an electrical load on biological tissues, but not biological load (since emulator or biological tissue is affected by generated voltage).

 

Application of this model as a calibration between pulse generators is a concern, since the electric pulse generators mainly vary in ability to give certain current with appropriate voltage. All other main value components depend on tissue parameters, individual placement if the electrodes and other. So, current or even generator dependant impedance calibration as such can be performed even with more simpler schemes in combination with commercially available devices (like impedance meter). However, I do believe that other applications might fit for this emulation system.

Can authors clarify the novelty of this paper? To my understanding, so far, this is the nice continuation of previous work (Langus et. Al. 2016). What is done, is the making of physical emulator and showing model with MatLab that practically repeats results which were already presented with finite element modelling (Langus et. Al. 2016).

Can authors explain why there is a difference of first pulse induced current in fig 2? To my understanding there is no difference in repetition frequency, as it is a first pulse that is presented.

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

Comments to Electronic Emulator of Biological load during electroporation

Authored by E. Pirc et al.,

 

Introduction: applied pulse amplitude has to specified in V/m and not in V as this quantity is not representative of the real electric fields experience by the tissues which depend on the electrode distance. May be both quantities can be mentioned: the electric field amplitudes and the applied voltages

 

Methods and Materials

Please specify briefly how current and voltage were monitored by Langus et al., 2016 this would add an interesting information to the paper together with the others already reported.

Procedure of down sampling and sample decimation for capacitive spikes and inductive/electroporation part of the pulse are not clear, try to better explain this procedure.

Furthermore the description of the numerical model in section 2.2.1 “Single monopolar pulse model” is not clear and it is difficult to exactly understand what has been done by the authors. They should try to rewrite this part having in mind that the readers should be able to replicate their work. Adding a scheme of the sequence of the procedure employed in the numerical model would surely help, with the inclusion the used options in the Matlab fitting tool box.

In the section 2.2.2 “Multiple monopolar pulse model” the computation of R4 from the LSE is not clear and again this procedure should be more clearly described as already said for previous section (a scheme would help in understating the exact procedure to be followed). Another important point to be described is the presence of the second diode in the circuit branch added to emulate multiple pulses at the higher repetition frequencies. Its role has not be discussed in the present text.

Author Response

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Author Response File: Author Response.pdf

Reviewer 3 Report

A case study on the design of an electronic emulator of biological load during electroporation is presented.

The objective is to create a systems capables to evaluate clinical, laboratory and prototype electroporation devices during the development process, or to evaluate their final performance considering at least from the perspective of output pulse parameters.

 

The proposed electronic load emulator is a proof of concept, which enables constant and sustainable testing and unbiased comparison of different electroporator’s operation that can be a very promising application. The proposed analog electrical circuit is obtained by exploiting literature available current and voltage measurements during electroporation of beef liver tissue ex vivo. Specific electrical networks are developped and the parameters of the corresponding analog circuit equations are derived. Single pulse and multiple pulses case are considered toghether with the effect of electoroporation memory (thanks to the use of inductive circuital element). These solution leads to take in to account the pulse delivery frequency dependency of the load current. Commercial available component are considered, except for the suitable realized magnetic one, and used to prototype and computer simulate (PSpice ORCAD enviromental) the proposed network. A good agrement with the experimental data, at least in a specific case of the three considered applied voltage, is showed.

 

The results and the apporach here presented are very interesting for the scientific community. However, some revision are suggested to the authors in order to better clarify methodological aspects, in particular in the subsection 2.2 - Circuital model

 

In the following a table presenting the proposed suggestion/modification in each specified row of the document with reported original authors’s text is reported

 

 

Nr.	Row document reference	Relative observed text	Suggestion/proposed modification
1	114, 138	Where	where
2	138-139	Where Vep represents applied electroporation pulse, with pulse voltage amplitude Uamp and Uth electroporation threshold voltage.	Vep is not in the equation. Please, reformulate the sentences
3	146-147	For numerical model, all measurement data (obtained and described earlier [19]), were imported into Matlab R2018b (MathWorks, Natick, Massachusetts, USA) computing environment, where values of optimal circuit elements were calculated.	The global optimal circuit elements calculation as to be better described. Could be useful to consider a flow-chart representation of the adopted optimization alghoritm. Moreover could be convenient to declare early that a deterministic optimization method is considered, define in a clear way the number and kind of parameters to optimize (complexity), their range of value (e.g by using a Table), and so on
4	149-152	The capacitive current spike part was due to its short duration - dynamics decimated by 10 and others by 70, though, in the end, the half of values represented a capacitive current spike and the other half an inductive/electroporation part of the pulse.	Not clear for me what you want to say
5	153	With the Matlab’s curve fitting toolbox	Please, report the fitting parameters adopted in the opitimization toolbox
6	158-163	Due to a large number of variables and three applied voltages, each variable range was predefined. …. While the threshold voltage was limited between 200 V and 400 V, because in Langus et. al. [19] optimal fitting maximum and minimum electric field magnitude limit were set to 20000 V/m and 40000 V/m. In this way all values of elements for single monopolar pulse circuit (Sch. 1) were determined.	This part could be used to better explain the reaserch method. The reader is confused by the order of the parameters identification. Is the Voltage threshold one of the parameter to optimize?
7	159-160	For resistive components and time constants, fifteen linearly distributed values between a minimal and maximal proposed optimal values from individual fit were used.	Please, report in the results section the considered range of parameters used to find the optimal solution
8	Eq. 5	t2	is this time-constant the same of the model adopted for monopulse case equations?
9	Eq. 6	t3	what is this parameter? Where is the parameter R4?
10	182	In	in
11	184-185	In this case, the whole sequence, all eight pulses were, included in the calculation.	Please, verify this sentence.
12	202	Because the time constant (329 us) of the circuit…	What is it? Where and how it is computed?
13	223	HZ	Hz
14		Sect. 3.1 Numerical calculation	Please, report in this section a Table with the estimated parameter used to derive the curves in Fig. 2a)

Comments for author File: Comments.pdf

Author Response

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Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Most of my concerns are cleared.

Please change:

Line 66-67 include that „some of the clinnically aproved electroporators significantly drop the voltage……”.

Also please check the references. Data like publishers, country are present in some references and in others it is absent.

Author Response

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Author Response File: Author Response.pdf