Brain Gene Silencing with Cationic Amino-Capped Poly(ethylene glycol) Polyplexes
, David Workman 1, Andreas G. Schätzlein 1,4 and Ijeoma F. Uchegbu 1,4,*Round 1
Reviewer 1 Report
Thank you for the authors for their valuable work. The Manuscript is well written in full details and presented clearly in a professional way.
I have only one comment regarding the nasal irritancy testing for the formulated nanoparticles.
Author Response
1. The formal nasal tolerability study has not yet been conducted and this has been stated in Lines 606 - 608.
Reviewer 2 Report
The manuscript «Brain gene silencing with cationic amino-capped poly(ethylene glycol) polyplex» submitted by Alamoudi A.A. et al. is devoted to the development of the relatively biocompatible vector for siRNA transfection into brain cells by intranasal administration (4APPA).Authors prepared siRNA – poly(ethylene glycol) and investigate the charge and size of resulted nanoparticles. They demonstrated relatively low cellular toxicity for polyplexes and possibility of cell association and uptake. Dawn regulation of ITCH gene was detected in the brain cells after intranasal dosing siRNA-4APPA nanoparticles.
I recommend accepting this manuscript for publication after minor revision.
I have some questions and remarks:
1) On figure 1 synthesis of tetramodified 4APEG-NH2 is presented, but authors obtained only one propylamine group per molecule. I think that this scheme contraries to obtained result and must be change.
2) Line 129 "mass" is missed before spectrometry, I think.
3) Lines 111 and 112. Please change the first letter in "Chloroform" from upper to ordinary size.
4) Lines 101, 273, 338 and 595. The point is absent in the end of sentences.
5) The rhodamine labeled cholesterol oxidase (line 256) used by authors to demonstrate encapsulation of the enzymes into vesicle, but there are no experimental details about preparation of this compound (RICT-ChOX). The methods of preparation and isolation or source of this compound must be added to the section Material and methods.
6) Please add the protocol of detection limit determination to the section Material and methods.
7) Line 178. Change uL for μL.
8) Please add all abbreviation interpretation (NuPAGE, ITCH…)
9) Please add the information about the fluorescently labelled siRNA purchasing.
10) Line 410. Please add the concrete panel for figure 2. I think it must be Figure 2a.
11) Table 1. Please check line 2. Is 4PPA correct?
12) Please add the designation of UT, NTC, FL and others abbreviations in the captures for the figures 2-5. What are the length in nucleotides for bands in marker on these figures?
13) I think that visualization of the results as diagrams can present your results more obvious than the number below the lanes.
14) Do you have any hypothesis why h9 targeting SMN H5A(+29+53) knockdowns full length SMN transcript levels?
15) How can you explain the presence of additional bands with lower mobility than full ength transcripts at the figure 2.
16) Are the sequences of recommended positive control exon skipping AOs unique in your work or the analogous sequences with other chemistries were used early in other publications?
17) I think that the controls those you recommended are very expensive for vide use in scientific research. Do you know the ways to reduce the cost of such controls?
Author Response
All changes to the manuscript appear in red font.
1. Figure 1 has been modified to illustrate the fact that not all of the amine groups were modified with propylamine groups.
2. Line 130 shows the brand name of the MALDI-TOF instrument and the word mass does not appear in this brand name. The trademark sign has been added to the brand name of the instrument for clarification in Line 130.
3. Chloroform has been corrected in Lines 111 and 112.
4. Full stop at the end of sentences in Lines 101, 273, 338 and 595. These have all been corrected where appropriate and was not found to be relevant in Line 101.
5. The comment on cholesterol oxidase does not appear to relate to this manuscript as cholesterol oxidase was not used in our studies.
6. The comment on the detection limit determination is not clear to us. We do not know to what this comment refers.
7. Line 178 - The mu sign has been corrected as requested.
8. The meaning of the ITCH abbreviation appeared in Line 157 of the original manuscript. It has been highligted in red. NuPAGE is a trade name for the gel used.
9. The source of the FAM-siRNA has been added to Lines 258-259.
10. In Line 411 - the reference to Figure 2 has been changed to read Figures 2a-2c.
11. Table 1 has been rectified on Page 11.
12. This comment on the need to add the terms UT, NTC and FL does not appear to refer to this manuscript and the terms UT, NTC and FL are unclear to us.
13. This comment on the visualisation of results is unclear to us. If this refers to the densiometry bar chart in Figure 3, then the gels is shown in Figure S8 of the Supplementary Information.
14. This comment on h9 targeting SMN does not appear to refer to our manuscript as we did not study this in our work.
15. This comment on the presence of additional bands in Figure 2 does not appear to refer to our manuscript as we do not have any bands in Figure 2.
16. This comment referring to exon skipping AOs does not appear to relate to our manuscript and we are unclear as to the meaning of AOs.
17. We are unsure on how to reduce the cost of our experiments and so do not have a valid response to this query.
