Simultaneous Down-Regulation of Intracellular hTERT and GPX4 mRNA Using MnO₂-Nanosheet Probes to Induce Cancer Cell Death

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Manuscript ID: sensors-4077669

Title: Simultaneous Down-regulation of Intracellular hTERT and GPX4 mRNA Using MnO₂-Nanosheet Probes to Induce Cancer Cell Death

Reviewer’s Comments

1) Apoptosis and ferroptosis are claimed but not mechanistically proven

The author(s) infer apoptosis/ferroptosis mainly from GSH depletion, ROS increase, and MDA elevation and from the general literature rationale. These markers are not sufficient to claim pathway-specific death.

Required additions (minimum):

• Apoptosis: Annexin V/PI (or TUNEL), caspase-3/7 activity, cleaved PARP/caspase-3 Western blot, and/or mitochondrial membrane potential (JC-1).
• Ferroptosis: BODIPY-C11 lipid ROS, GPX4 protein (WB), and rescue experiments using ferrostatin-1/liproxstatin-1 (and ideally deferoxamine).
• A pathway dissection should show: apoptosis inhibitors (e.g., z-VAD-fmk) partially rescue; ferroptosis inhibitors partially rescue; combined rescue is strongest.

Without inhibitor rescue + pathway markers, the central conclusion (“dual apoptosis and ferroptosis”) remains overstated.

 

2) “Synergy” is asserted, but synergy is not quantitatively demonstrated

The manuscript repeatedly uses “synergistic” language (e.g., dual-hit strategy; synergy between apoptosis and ferroptosis). Yet, cell viability data are shown across groups, without a formal synergy model.

Required:

• Quantify synergy using Bliss independence, Loewe additivity, HSA, or Chou–Talalay combination index.
• Include proper comparators:
• Dual-loaded probe vs (Anti-hTERT-MnO₂-NS + Anti-GPX4-MnO₂-NS) co-treatment at matched doses
• Dual-loaded probe vs free ASOs (with/without MnO₂-NS)
  The current text notes single-target probes and dual probes, but not a rigorous synergy analysis.

 

3) Target engagement is incomplete: mRNA ↓ shown, but protein-level GPX4 is not directly measured

The author(s) measured hTERT via ELISA and used “Se-GPX activity” as a proxy for GPX4. This is problematic because “Se-GPX activity” reflects multiple selenoprotein GPXs, not uniquely GPX4.

Required:

• Measure GPX4 protein directly (Western blot or validated GPX4 ELISA).
• Confirm that the ASO reduces GPX4 selectively, not just overall antioxidant enzyme activity.
• Provide knockdown magnitude (%, with error bars) and link it to functional ferroptosis markers.

 

4) No evidence that the antisense strands act through sequence-specific antisense mechanisms (specificity/off-target)

The study relies on fluorescent “Anti-hTERT-DNA” and “Anti-GPX4-DNA” strands (Table 1) but lacks key ASO validation elements:

Required:

• Clarify whether these are DNA ASOs, whether they are phosphorothioate-modified, 2′-O-Me/LNA, etc. (critical for stability and RNase-H recruitment).
• Include a scrambled ASO control for each target (not only a single “Control-DNA”) and/or mismatch ASO control.
• Perform RNase-H dependence (if that is the claimed mechanism) or provide rationale if steric-block strategy is used.
• Add at least one off-target screen: e.g., measure unrelated transcripts, or include RNA-seq/target panel.

Right now, the control condition primarily tests MnO₂-NS + a control strand, not robust antisense specificity.

5) Nanomaterial/probe characterization is insufficient for reproducibility and biological interpretation

Characterization presented includes morphology and a very broad DLS range (50–600 nm), plus UV–vis peak around 370 nm. This is not enough.

Required:

• Report mean hydrodynamic diameter ± SD, PDI, and zeta potential (before/after loading).
• Provide loading efficiency/capacity for each ASO (e.g., nmol per mg MnO₂-NS), and batch-to-batch variability.
• Provide stability in serum/complete media (aggregation, size over time).
• Provide GSH-triggered release kinetics (time course), not just end-point recovery with different GSH concentrations.
• Measure Mn²⁺ release and/or MnO₂ consumption in relevant conditions (intracellular mimic).

 

6) Biological scope is limited: no normal cell toxicity/selectivity

The author(s) test four cancer cell lines for uptake/viability (A549, HeLa, HepG2, Caco-2) but there is no normal cell line control to evaluate therapeutic window.

Required:

• Include at least one non-malignant cell line (e.g., normal epithelial or fibroblast) with matched dosing to claim “anticancer” selectivity.
• Provide selectivity metrics (IC50 cancer vs normal).

 

7) Confocal uptake data are qualitative; need quantification and localization controls

CLSM images show dual fluorescence after 6 h, but there is no quantification.

Required:

• Quantify uptake (flow cytometry mean fluorescence intensity for FAM and Cy5).
• Add subcellular localization (lysosomal colocalization; endosomal escape evidence).
• Demonstrate that fluorescence recovery corresponds to intracellular release (not surface-bound signal).

 

8) Statistical reporting is inconsistent and under-specified

Statistical methods state significance at P<0.05 (*) and “high significance” at P<0.01 (**), yet figures include ***P<0.001 and Figure 5 caption uses ***P<0.001.

 

Required:

• Specify exact tests used (t-test? one-way ANOVA with post-hoc? multiple comparisons correction?).
• Define *, **, *** consistently across manuscript.
• Report n (biological replicates) per assay, not just “at least three”.

 

9) Methodological errors/ambiguities that affect reproducibility

• Section 2.9 says cells were treated “as described in Section 2.9,” which is self-referential and likely incorrect; it should reference the relevant prior treatment section.

 

• For qRT-PCR: The author(s) must report ΔΔCt method, primer efficiencies, melt curves, and normalization details (GAPDH is listed but method is not fully described).

 

• Probe concentrations are described (e.g., 30 µg/mL), but the actual ASO dose delivered per well is not reported; this is critical to compare with literature.

10) Interpretation overreach in Results/Discussion

The discussion often implies causality (dual apoptosis/ferroptosis) from correlative oxidative stress markers. Please soften language unless mechanistic validation is added.

Comments for author File: Comments.pdf

Author Response

Reviewer 1

 

Manuscript ID: sensors-4077669

Title: Simultaneous Down-regulation of Intracellular hTERT and GPX4 mRNA Using MnO₂-Nanosheet Probes to Induce Cancer Cell Death

 

Reviewer’s Comments

• Apoptosis and ferroptosis are claimed but not mechanistically proven

The author(s) infer apoptosis/ferroptosis mainly from GSH depletion, ROS increase, and MDA elevation and from the general literature rationale. These markers are not sufficient to claim pathway-specific death.

Required additions (minimum):

. Apoptosis: Annexin V/PI (or TUNEL), caspase-3/7 activity, cleaved PARP/caspase-3 Western blot, and/or mitochondrial membrane potential (JC-1).

. Ferroptosis: BODIPY-C11 lipid ROS, GPX4 protein (WB), and rescue experiments using ferrostatin-1/liproxstatin-1 (and ideally deferoxamine).

. A pathway dissection should show: apoptosis inhibitors (e.g., z-VAD-fmk) partially rescue; ferroptosis inhibitors partially rescue; combined rescue is strongest.

Without inhibitor rescue + pathway markers, the central conclusion (“dual apoptosis and ferroptosis”) remains overstated.

 

Response: We sincerely thank the reviewer’s suggestion. The present study aims to design a simple system for preliminary validation of the combined effect of simultaneously downregulating hTERT and GPX4 using antisense oligonucleotide technology, in synergy with the reduction of intracellular GSH induced by manganese dioxide, on killing cancer cells. However, a more comprehensive study of this system would indeed require detail analysis of the pathway of apoptosis and ferroptosis. These investigations will be further carried out in future in vivo anti-cancer research.

 

 

• “Synergy” is asserted, but synergy is not quantitatively demonstrated

The manuscript repeatedly uses “synergistic” language (e.g., dual-hit strategy; synergy between apoptosis and ferroptosis). Yet, cell viability data are shown across groups, without a formal synergy model.

Required:

. Quantify synergy using Bliss independence, Loewe additivity, HSA, or Chou–Talalay combination index.

.  Include proper comparators:

o Dual-loaded probe vs (Anti-hTERT-MnO₂-NS + Anti-GPX4-MnO₂-NS) co-treatment at matched doses.

o  Dual-loaded probe vs free ASOs (with/without MnO₂-NS).

The current text notes single-target probes and dual probes, but not a rigorous synergy analysis.

 

Response: We sincerely thank the reviewer’s suggestion. The work presented here represents only a preliminary exploration into the induction of cancer cell death via the simultaneous downregulation of hTERT and GPX4 using antisense technology. The experimental design and data analysis were carried out with reference to relevant literature. In response to the reviewer's suggestion regarding quantifying synergy using methods such as Bliss independence, Loewe additivity, HSA, or the Chou–Talalay combination index, we plan to gradually conduct detailed and comprehensive studies on these aspects in the future. As for the reviewer's proposal to perform control experiments using free ASOs without MnO₂-NS, we consider it unnecessary. It is well known that in the absence of a delivery carrier, free ASOs cannot cross the cell membrane to enter the cells, and therefore, cell death would not be induced.

 

 

• Target engagement is incomplete: mRNA ↓ shown, but protein-level GPX4 is not directly measured

The author(s) measured hTERT via ELISA and used “Se-GPX activity” as a proxy for GPX4. This is problematic because “Se-GPX activity” reflects multiple selenoprotein GPXs, not uniquely GPX4.

Required:

. Measure GPX4 protein directly (Western blot or validated GPX4 ELISA).

. Confirm that the ASO reduces GPX4 selectively, not just overall antioxidant enzyme activity.

. Provide knockdown magnitude (%, with error bars) and link it to functional ferroptosis markers.

 

Response: We sincerely thank the reviewer’s suggestion. The GPX4 activity in the cell lysates has been determined using a commercial GPX4 Fluorogenic Assay Kit. The results confirmed that the ASO reduces GPX4 selectively.

 

• No evidence that the antisense strands act through sequence-specific antisense mechanisms (specificity/off-target)

The study relies on fluorescent “Anti-hTERT-DNA” and “Anti-GPX4-DNA” strands (Table

1) but lacks key ASO validation elements:

Required:

. Clarify whether these are DNAASOs, whether they are phosphorothioate-modified, 2′-O-Me/LNA, etc. (critical for stability and RNase-H recruitment).

. Include a scrambled ASO control for each target (not only a single “Control-DNA”) and/or mismatch ASO control.

. Perform RNase-H dependence (if that is the claimed mechanism) or provide rationale if steric-block strategy is used.

. Add at least one off-target screen: e.g., measure unrelated transcripts, or include RNA-seq/target panel.

Right now, the control condition primarily tests MnO₂-NS + a control strand, not robust antisense specificity.

 

Response: We sincerely thank the reviewer’s suggestion. This study is a continuation of our previous work. The two antisense sequences targeting hTERT mRNA and GPX4 mRNA used here are the same as those employed in prior studies, and their effectiveness has been validated in earlier research. Further investigations will be progressively carried out in future work.

 

5) Nanomaterial/probe characterization is insufficient for reproducibility and biological interpretation

Characterization presented includes morphology and a very broad DLS range (50–600 nm), plus UV–vis peak around 370 nm. This is not enough.

Required:

. Report mean hydrodynamic diameter ± SD, PDI, and zeta potential (before/after loading).

. Provide loading efficiency/capacity for each ASO (e.g., nmol per mg MnO₂-NS), and batch-to-batch variability.

. Provide stability in serum/complete media (aggregation, size over time).

. Provide GSH-triggered release kinetics (time course), not just end-point recovery with different GSH concentrations.

. Measure Mn²⁺ release and/or MnO₂ consumption in relevant conditions (intracellular mimic).

 

Response: We sincerely thank the reviewer’s suggestion. In accordance with the reviewers' comments, we have conducted additional relevant experiments and incorporated the findings into the manuscript. Nevertheless, due to time limitations, we regret that we could not perform all the supplementary experiments as requested. 

 

• Biological scope is limited: no normal cell toxicity/selectivity

The author(s) test four cancer cell lines for uptake/viability (A549, HeLa, HepG2, Caco-2) but there is no normal cell line control to evaluate therapeutic window.

Required:

. Include at least one non-malignant cell line (e.g., normal epithelial or fibroblast) with matched dosing to claim “anticancer” selectivity.

. Provide selectivity metrics (IC50 cancer vs normal).

 

Response: We sincerely thank the reviewer’s suggestion. We evaluated the cytotoxicity of this system against normal hepatocytes HL7702. The results showed that its cellkilling effect on normal cells was significantly weaker than that on cancer cells. Moreover, the cytotoxicity of the three antisenseloaded manganese dioxide probes was comparable to that of the control probe. Given the low expression of hTERT and GPX4 as well as the lower GSH content in HL7702 cells, it is inferred that the system exerts specific cytotoxicity toward cancer cells.

 

 

• Confocal uptake data are qualitative; need quantification and localization controls

CLSM images show dual fluorescence after 6 h, but there is no quantification.

Required:

. Quantify uptake (flow cytometry mean fluorescence intensity for FAM and Cy5).

. Add subcellular localization (lysosomal colocalization; endosomal escape evidence).

. Demonstrate that fluorescence recovery corresponds to intracellular release (not surface-bound signal).

 

Response: We sincerely thank the reviewer’s suggestion. The present study aims to design a simple system for preliminary validation of the combined effect of simultaneously downregulating hTERT and GPX4 using antisense oligonucleotide technology, in synergy with the reduction of intracellular GSH induced by manganese dioxide, on killing cancer cells. The quantification and localization controls investigations will be further carried out in future research.

 

 

8) Statistical reporting is inconsistent and under-specified

Statistical methods state significance at P<0.05 (*) and “high significance” at P<0.01 (**), yet figures include ***P<0.001 and Figure 5 caption uses ***P<0.001.

Required:

. Specify exact tests used (t-test? one-way ANOVA with post-hoc? multiple comparisons correction?).

. Define *, **, *** consistently across manuscript.

. Report n (biological replicates) per assay, not just “at least three” . 

 

Response: We sincerely thank the reviewer’s comments. The statistical reporting has been revised across manuscript.

 

• Methodological errors/ambiguities that affect reproducibility

. Section 2.9 says cells were treated “as described in Section 2.9,” which is self-

referential and likely incorrect; it should reference the relevant prior treatment section.

. For qRT-PCR: The author(s) must report ΔΔCt method, primer efficiencies, melt

curves, and normalization details (GAPDH is listed but method is not fully described).

. Probe concentrations are described (e.g., 30 µg/mL), but the actual ASO dose delivered per well is not reported; this is critical to compare with literature.

 

Response: We sincerely thank the reviewer’s comments. Here we really made a typing mistake. “as described in Section 2.9” has been corrected into “as described in Section 2.8”. For qRT-PCR, the detailed experimental method has been given in the Section 2.9 and labeled as red. Nevertheless, due to time limitations, we regret that we could not perform all the supplementary experiments as requested. 

 

• Interpretation overreach in Results/Discussion

The discussion often implies causality (dual apoptosis/ferroptosis) from correlative oxidative stress markers. Please soften language unless mechanistic validation is added.

Response: We sincerely thank the reviewer’s suggestion. We have made revisions in the relevant discussions in the text, primarily focusing on the experimental results and avoiding elaboration on the synergistic effects of apoptosis and ferroptosis in the system we prepared.

Reviewer 2 Report

Comments and Suggestions for Authors

This paper focuses on the field of cancer precision therapy and designs a manganese dioxide nanosheet-based co-delivery probe, which can simultaneously target hTERT and GPX4 mRNA and downregulate their expression. Through the synergistic effect of glutathione depletion and gene silencing, the probe induces dual pathways of apoptosis and ferroptosis in cancer cells, thereby providing a novel strategy for multifunctional nanotherapy. However, there are still some issues that require modification.

1.There are some minor errors in the manuscript. For example, the phrase "in a humidified atmosphere of 5% CO" on Page 4, Line 166 should be corrected to "in a humidified atmosphere of 5% CO₂。

2.The formatting of the references is inconsistent and requires revision.

3.The discussion section needs to be strengthened by comparison with existing studies: it is recommended to add a comparative analysis of this study with other hTERT/GPX4-targeted therapies or MnO₂-based nanomedicines , so as to clarify the advantages and limitations of this study.

4.It is recommended that the authors further discuss the potential challenges of the probe for in vivo applications and propose corresponding solutions.

5.The synergistic ratio of apoptosis and ferroptosis needs to be quantified: the manuscript confirms the occurrence of dual cell death, but fails to clarify the proportion of apoptosis and ferroptosis in cancer cell death. It is recommended to quantify the contribution of the two death pathways through blocking experiments combining flow cytometry with a ferroptosis inhibitor  and an apoptosis inhibitor , so as to further highlight the synergistic effect.

6. The current experiments only focus on cancer cell viability and fail to detect the probe's effects on normal cells. It is recommended to supplement the toxicity data of normal cells to evaluate the biosafety of the probe and enhance the persuasiveness of its clinical translation.

Author Response

Reviewer 2

 This paper focuses on the field of cancer precision therapy and designs a manganese dioxide nanosheet-based co-delivery probe, which can simultaneously target hTERT and GPX4 mRNA and downregulate their expression. Through the synergistic effect of glutathione depletion and gene silencing, the probe induces dual pathways of apoptosis and ferroptosis in cancer cells, thereby providing a novel strategy for multifunctional nanotherapy. However, there are still some issues that require modification.

1. There are some minor errors in the manuscript. For example, the phrase "in a humidified atmosphere of 5% CO" on Page 4, Line 166 should be corrected to "in a humidified atmosphere of 5% CO₂。

Response: We sincerely thank the reviewer’s comment. We have corrected the typing here. 

 

2. The formatting of the references is inconsistent and requires revision.

Response: We sincerely thank the reviewer’s suggestion. All references have been reformatted to meet the Sensors journal template requirements, and the corrections has been labeled as red.

 

3. The discussion section needs to be strengthened by comparison with existing studies: it is recommended to add a comparative analysis of this study with other hTERT/GPX4-targeted therapies or MnO₂-based nanomedicines, so as to clarify the advantages and limitations of this study.

Response: We sincerely thank the reviewer’s comment. We have added a comparative analysis between this work and previously reported studies in the text. Overall, the greatest advantage of the Anti-hTERT/GPX4-MnO₂-NS probes designed here lies in their simple preparation and high efficiency in promoting cell death. Compared with many previously reported hTERT/GPX4-targeted therapies or MnO₂-based nanomedicines, it has been found that achieving the same level of cell death induction often requires more complex drug system preparation processes or highly intricate functional components, leading to lower reproducibility across different sample batches and higher preparation costs. In contrast, simpler methods or systems exhibit lower efficiency in promoting cell death and fail to achieve effective anticancer outcomes. However, numerous challenges remain when applying this system for in vivo anticancer therapy, such as tumor targeting and the leakage of the drug during systemic circulation. Therefore, there is considerable room for optimization of the system. For instance, modifying the surface of manganese dioxide nanosheets with hyaluronic acid, a tumortargeting component, could enhance tumorspecific targeting and reduce the leakage of bare manganese dioxide nanosheets during circulation. The specific effects of such modifications will be further investigated in subsequent studies.

 

4. It is recommended that the authors further discuss the potential challenges of the probe for in vivo applications and propose corresponding solutions.

Response: We sincerely thank the reviewer’s suggestion. The present study aims to design a simple system for preliminary validation of the combined effect of simultaneously downregulating hTERT and GPX4 using antisense oligonucleotide technology, in synergy with the reduction of intracellular GSH induced by manganese dioxide, on killing cancer cells. However, numerous challenges remain when applying this system for in vivo anticancer therapy, such as tumor targeting and the leakage of the drug during systemic circulation. Therefore, there is considerable room for optimization of the system. For instance, modifying the surface of manganese dioxide nanosheets with hyaluronic acid, a tumortargeting component, could enhance tumorspecific targeting and reduce the leakage of bare manganese dioxide nanosheets during circulation. The specific effects of such modifications will be further investigated in subsequent studies.

 

5. The synergistic ratio of apoptosis and ferroptosis needs to be quantified: the manuscript confirms the occurrence of dual cell death, but fails to clarify the proportion of apoptosis and ferroptosis in cancer cell death. It is recommended to quantify the contribution of the two death pathways through blocking experiments combining flow cytometry with a ferroptosis inhibitor and an apoptosis inhibitor , so as to further highlight the synergistic effect.

Response: We sincerely thank the reviewer’s suggestion. The present study aims to design a simple system for preliminary validation of the combined effect of simultaneously downregulating hTERT and GPX4 using antisense oligonucleotide technology, in synergy with the reduction of intracellular GSH induced by manganese dioxide, on killing cancer cells. However, a more comprehensive study of this system would indeed require quantitative analysis of the proportions of apoptosis and ferroptosis. These investigations will be further carried out in future in vivo anti-cancer research.

 

6. The current experiments only focus on cancer cell viability and fail to detect the probe's effects on normal cells. It is recommended to supplement the toxicity data of normal cells to evaluate the biosafety of the probe and enhance the persuasiveness of its clinical translation.

 

Response: We sincerely thank the reviewer’s suggestion. We evaluated the cytotoxicity of this system against normal hepatocytes HL-7702. The results showed that its cell-killing effect on normal cells was significantly weaker than that on cancer cells. Moreover, the cytotoxicity of the three antisense-loaded manganese dioxide probes was comparable to that of the control probe. Given the low expression of hTERT and GPX4 as well as the lower GSH content in HL-7702 cells, it is inferred that the system exerts specific cytotoxicity toward cancer cells.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have provided clear, thorough, and well-justified responses to all reviewer comments. The revisions have been carefully implemented and have substantially improved the clarity, scientific rigor, and overall quality of the manuscript. In its current form, the manuscript meets the journal’s publication standards and is therefore accepted for publication.

Reviewer 2 Report

Comments and Suggestions for Authors

The author has provided detailed responses to the reviewers' comments and made corresponding revisions, and it is recommended to accept the paper.