Cancer Stem Cell Target Labeling and Efficient Growth Inhibition of CD133 and PD-L1 Monoclonal Antibodies Double Conjugated with Luminescent Rare-Earth Tb³⁺ Nanorods

Round 1

Reviewer 1 Report

Dear Editor in chief:

Applied sciences Journal

Regarding Manuscript: applsci-714379

``Cancer stem cell target labeling and efficient growth inhibition of nanocomplex-a double conjugated CD133 and PD-L1 monoclonal antibodies with Tb3+ luminescent rare-earth`` by Thi Thao Do and coworkers:

Authors have produced a nanocomplex that is conjugated with two monoclonal antibodies against CD133 and PD-L1 using nanomaterial TbPO4.H2O and examined it using NTERA-2 CSC cell line that overexpress CD133 and PD-L1 and examined the effects of their nanocomplex on this cell line.

 Overall, this manuscript has been written well with new and interesting findings. However, I suggest authors to:

Authors have used a cell with high expression of CD133 and PD-L1. Have they used other cell lines with medium or low expression of these 2 markers and test the cytotoxic effects of TMC? Extend the figure legend description. Therefore, readers can find the results after reading the legends. Authors have used 2 experiments to show the effects of TMC on tumor cells. It might be better to use other assays such as wound healing or migration assays for more validation.

Minor comments

Check the spell of words. E.g. PL-D1 that should be PD-L1 in abstract. Check the whole manuscript. For each reagent in MM section, writhe the Company name, city and country of production.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

The research addressed in the manuscript entitled "Cancer stem cell target labeling and efficient growth inhibition of the nanocomplex - a double conjugated CD133 and PD-L1 antibodies with Tb³⁺ luminescent rare-earth" is of great interest in the field of cancer therapy. it can be considered for publication in the Journal Applied Sciences, but with considerable changes.

I recommend the authors to improve their manuscript and re-submit it to the journal. Some of the comments on the text are:

What was the pH value of the solution before adjusting it to 2 by adding 10% NaOH solution, in the synthesis of TbPO₄.H₂O. It would be more clear if the authors will indicate the TbPO4:TEOS ratio. The functionalization degree should be mentioned in the text. As well here: "The glycerol solution was added to a hydrous mixture of ethanol containing TbPO4·H2O coated silica (TbPO4·H2O@silica)". How much glycerol? To how much TbPO₄? The same comment here: "3-Aminopropyl trimethoxy silane (APTMS) was dispersed in ethanol before mixed with TbPO4·H2O@ silica solution and stirred for 15 minutes to functionalize the surface with -NH2". Again, How much APTMS was dispersed in what quantity of ethanol? What is the TbPO₄·H₂O@ silica:APTMS ratio? "...and finally dissolving in PBS" - are sure the authors the solid material has been dissolved in PBS? Also in the 2.2 section the authors should mention the ration between the used synthesized solid and monoclonal antibodies. Line 124: "labling" should be corrected. Nowhere in the manuscript could be find the meaning of "mAb-FITC". It is very hard to observe from SEM images that the nanorod diameter after mAbs conjugating on the surface. In the Figure 3 could not be find the fluorescence spectrum of TbPO₄·H₂O @ silica-NH₂ sample and IR spectrum of TbPO₄.H₂O @silica-NH₂-mAb sample. The axis names should be rewritten! The comments on the figure 3 should be improved. More discussions are necessary. For example, the bond appearance as result of transmission peak from IR spectrum, and so on. As well, a comparison with the fluorescence spectrum of the used reference should be discussed. The manuscript should be be carefully checked for English and edited in compliance with the rules for writing chemical formulas.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

In this study, the authors produced a double conjugated CD133 and anti-PD-L1 monoclonal antibodies with nanomaterial TbPO4.H2O in order to investigate effectively targeting cancer stem cells. The detection of CRCs using the CD133 marker or the increase of T-cell response for antitumor effect using the anti-PD-L1 antibody is a well-known theory. Although research using well-known markers, it is very interesting that  CD133 and anti-PD-L1 are conjugated to produce material by nanotechnology that can be used simultaneously. Please address the following comments on errors or revision points that may be raised through the contents
1. In addition to cancer stem cells, CD133 is a useful marker that can detect the expression of stem-like cells throughout the body. Therefore, it would be considered that specifically targeting cancer stem cells is inefficient when the nanoparticles were used in clinical.
2. There are also cancer stem cells that do not express CD133 among various cancer cells (CD133 negative cancer stem cells). Single experimentation with human embryonal carcinoma cell lines is insufficient to determine the effectiveness of this nanoparticle.
3. It does not give any theoretical background on the Terbi mentioned abstract. The nanomaterial TbPO4.H2O likewise has no theoretical background.
4. Figure 4d shows green fluorescence despite the group of the negative control. And, it seems that In addition, CD133-FITC shows better fluorescence detection than the conjugated nanoparticles. Insert a scale bar in each figure.
5. In Figure 6, CCD-18Co is the normal type fibroblast of the colon. The scientific rationale for the use of colon fibroblast could not be derived while human embryonic carcinoma was used in the main study. Insert a scale bar in each figure.
6. In Table 2, this result could not conclude that there was a significant difference. Moreover, even if the PD-L1 was inhibited by the nanoparticle, cell death must be caused by immune cytotoxicity.
7. This reviewer wonders what the meaning of the results in figure 7 is.
8. The photo presented in figure 8 is not a spheroid 3d culture, it is considered as a colony-forming assay. The most awkward in figure 8 is that it was predicted the attack of cancer cells by macrophages, but it did not. The author must present the activity of Macrophage. And why is the negative control group excluded from the result in figure 8?
9. In whole content, no statistical significance is found in the presented results.
10. Since cancer stem cells grow more aggressively in relatively low oxygen, it is best to perform the study under hypoxic condition instead of the normoxic condition.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 4 Report

The manuscript “Cancer stem cell target labeling and efficient growth inhibition of nanocomplex-a double conjugated CD133 and PD-L1 monoclonal antibodies with tb³⁺ luminescent rare-earth” submitted by Thi Thao Do and coworkers proposed a new nanostructured material conjugated with anti CD133 and PD-L1 monoclonal antibodies and Tb3+ (termed as TMC) for its application on cancer therapy. The manuscript provides evidence of the physicochemical characterization of such novel material and its cytotoxic effects against two different cell lines, NTERA-2 and CCD-18Co. In addition, the authors also evaluated the effects of TMC against NTERA-2 spheroids when co-cultured in vitro with macrophages. Overall, the paper is well-written and supplies relevant information on the synthesis and mechanism of this novel nanostructured material and that can be interesting for the field. However, I several section should be improved and I strongly recommend the incorporation of additional data to improve the final quality of the work.

 

Major comments:

 

My major concern is regarding the stability of the new nanomaterial. Please provide experimental data in order to demonstrate the stability and potential release of the conjugated antibodies under physiological conditions over time. The amount/s of functional antibodies present in TMC should be experimentally demonstrated and compared with the corresponding free antibodies. The process of Ab coupling to the nanomaterial might induce denaturation and/or aggregation of the ab, which may lead to a loss of function. This should be also properly discussed in the manuscript. The authors mention in lines 132-141 that “cells were seeded in 96 well plates and treated with TMC at various concentrations for 72 hours…”. Please complete this section with all the necessary details and show the corresponding dose-response data in the manuscript. I strongly recommend the calculation of the corresponding IC50 values. This should be also compared with the values of the IC50 values with equivalent doses of the free antibodies for comparative purposes. The effect of the free antibodies against the NTERA-2 spheroids should be evaluated and compared with TMC. This should be properly discussed in the revised version of the manuscript.    Discussion section, lines 233-254. The authors mentioned here: “Together with CSC probing, TMC with PD-L1 mAb in structure were produced for the puropose of cancer treatment.”. Please explain and discuss the main therapeutic advantages of this novel material compared with the free anti- PD-L1 or anti- CD133 ab. The following relevant reverence should be included: Nanoscale. 2017 May 11;9(18):611-6121. doi:10.1039/c7nr00947j.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

The manuscript has been revised according reviewers comments and can be considered for publication in the Journal of Applied Sciences.

Reviewer 3 Report

All concerns raised by this reviewer have been well addressed.

Reviewer 4 Report

The authors have revised the manuscript and corrected all errors and drawbacks present in the initial version. I recommend the publication in present form.