Title: Effect of chitosan nanoparticles conjugated with the cell free supernatant of
Bifidobacterium bifidum on the expression of genes related to colorectal cancer in colon
adenocarcinoma (Caco-2) cell line
Version: 0 Date: 03 Nov 2024
Reviewer's report:
In this manuscript, the authors reported on a culture supernatant of Bifidobacterium bifidum-
loaded chitosan nanoparticles and used it to evaluate its effect on gene expression in the colon
adenocarcinoma (Caco-2) cell line. Particle size, zeta potential and morphology as well as
protein release of the nanoparticles were analysed. The gene expression levels after treatment
were also analyzed. This study reports some interesting findings, but there are many important
concerns that need to be carefully considered. 1) The characterization of nanoparticles is not
clear, e.g, PDI should be provided; TEM is required to characteize the surface morphology
of nanoparticles. 2) The protein loading content should be calculated, as it is more important
than  the loading efficiency.  3) The preparation of the nanoparticles appears to be based on
electrostatic or ionic interactions, so that the prepared nanoparticles theoretically dissociate
rapidly and release a large number of proteins in an acidic environment at pH 1.8. 4#The authors
need to further explain why the concentration of CTNP in cell experiments is 0.05%; B.b-sup
chose 1200 µg. 5) What is "MSC CM", the authors should carefully check all similar errors.  6#In
addition, the authors need to explain why Caco-2 cells were chosen; actually, this kind of cell
line has been widely used for evaluating the intestinal mucosal barrier integrity, e.g.,  10.1002/
advs.202308092; 10.1002/adfm.202304381. 7) The authors need to find out as much as possible
about which proteins in the supernatant play a role in regulating gene expression.
Title: Effect of chitosan nanoparticles conjugated with the cell free supernatant of
Bifidobacterium bifidum on the expression of genes related to colorectal cancer in colon
adenocarcinoma (Caco-2) cell line
Version: 0 Date: 03 Nov 2024
Reviewer's report:
In this manuscript, the authors reported on a culture supernatant of Bifidobacterium bifidum-
loaded chitosan nanoparticles and used it to evaluate its effect on gene expression in the colon
adenocarcinoma (Caco-2) cell line. Particle size, zeta potential and morphology as well as
protein release of the nanoparticles were analysed. The gene expression levels after treatment
were also analyzed. This study reports some interesting findings, but there are many important
concerns that need to be carefully considered. 1) The characterization of nanoparticles is not
clear, e.g, PDI should be provided; TEM is required to characteize the surface morphology
of nanoparticles. 2) The protein loading content should be calculated, as it is more important
than  the loading efficiency.  3) The preparation of the nanoparticles appears to be based on
electrostatic or ionic interactions, so that the prepared nanoparticles theoretically dissociate
rapidly and release a large number of proteins in an acidic environment at pH 1.8. 4#The authors
need to further explain why the concentration of CTNP in cell experiments is 0.05%; B.b-sup
chose 1200 µg. 5) What is "MSC CM", the authors should carefully check all similar errors.  6#In
addition, the authors need to explain why Caco-2 cells were chosen; actually, this kind of cell
line has been widely used for evaluating the intestinal mucosal barrier integrity, e.g.,  10.1002/
advs.202308092; 10.1002/adfm.202304381. 7) The authors need to find out as much as possible
about which proteins in the supernatant play a role in regulating gene expression.
Please review this paper.
user
Title: Effect of chitosan nanoparticles conjugated with the cell free supernatant of
Bifidobacterium bifidum on the expression of genes related to colorectal cancer in colon
adenocarcinoma (Caco-2) cell line
Version: 0 Date: 03 Nov 2024
Reviewer's report:
In this manuscript, the authors reported on a culture supernatant of Bifidobacterium bifidum-
loaded chitosan nanoparticles and used it to evaluate its effect on gene expression in the colon
adenocarcinoma (Caco-2) cell line. Particle size, zeta potential and morphology as well as
protein release of the nanoparticles were analysed. The gene expression levels after treatment
were also analyzed. This study reports some interesting findings, but there are many important
concerns that need to be carefully considered. 1) The characterization of nanoparticles is not
clear, e.g, PDI should be provided; TEM is required to characteize the surface morphology
of nanoparticles. 2) The protein loading content should be calculated, as it is more important
than  the loading efficiency.  3) The preparation of the nanoparticles appears to be based on
electrostatic or ionic interactions, so that the prepared nanoparticles theoretically dissociate
rapidly and release a large number of proteins in an acidic environment at pH 1.8. 4#The authors
need to further explain why the concentration of CTNP in cell experiments is 0.05%; B.b-sup
chose 1200 µg. 5) What is "MSC CM", the authors should carefully check all similar errors.  6#In
addition, the authors need to explain why Caco-2 cells were chosen; actually, this kind of cell
line has been widely used for evaluating the intestinal mucosal barrier integrity, e.g.,  10.1002/
advs.202308092; 10.1002/adfm.202304381. 7) The authors need to find out as much as possible
about which proteins in the supernatant play a role in regulating gene expression.
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