In the manuscript entitled Replication of “Carbon-Dot-Based Dual-Emission Nanohybrid Produces a Ratiometric Fluorescent Sensor for In Vivo Imaging of Cellular Copper Ions” the authors aimed at replicating a study on the use of carbon dots for intracellular sensing of Cu2+, performing additional experiments in order to assess if a high proportion of the nanoparticles escape the endosomes and to detect copper ions in a cell model of the Wilson’s disease.

The methodology is well detailed, rigorous and will allow reaching the objectives of the replication study, in my opinion. In addition, appropriate controls will be performed in order to support the results.

I have a few comments and questions:

- Has the synthesis of CdSe@C-TPEA been reproduced in other articles (among the > 474 articles that cited the original article)?

- In the original work, the size of the CQDs assessed from HRTEM images is around 5 nm (the number of measurements was not mentioned). Measuring the size of CQDs by HRTEM is very challenging. Hence, I think the variation of maximum 10% (mentioned in the table) might be too strict. What do the authors plan to do if the measurements are not within 10% of the average size value? Similar comments for the Cu2+-dependent fluorescence of CdSe@C-TPEA if r2< 0.9.

- How did the author chose the value of 20% as ”threshold” to state that a high proportion of CdSe@C-TPEA escape endosomes?

- The authors propose to use quinine sulfate as a standard to assess the quantum yield of the CQDs, while rhodamine B was used in the original work, in order to more precisely determine the quantum yield. In parallel, it would be also useful to use rhodamine B to see if the quantum yield is in agreement with the value reported in the original article.

- The NMR spectroscopy confirmed the structure of all synthesized molecules. Do the authors know what is the broad peak at around 1.75 ppm in the NMR spectrum of CPD1? It does not seem to correspond to a solvent residual peak, does it?

- Do the authors plan to study the photostability of CdSe@C-TPEA?

List of minor corrections to improve the readability of the manuscript:

- Some abbreviations are not defined.

- BOC should be corrected to Boc.

- Scheme 2, 4 & 5: add the solvent in the conditions below the arrow (even if it is mentioned in the protocols), otherwise indicating “Reflux” is meaningless.

- Section 1.9: correct “were added” to “will be added”. Correct “Cus” by “Cys” and also “Phy” by “Phe” (“Phy” appears in the x axis of Fig. 3b in the original paper, but I guess it was a mistake too). Correct “Iso” by “Ile” (the three letter code of isoleucine is Ile and not Iso).

- Add the exact reference in the section 1.14.4.

Reproducibility in nanoscience is of great importance because small variations in sample parameters or measurements can significantly impact the results and conclusions. Ensuring the reliability and validity of scientific findings in nanoscience, as well as the implementation of newly developed and validated protocols as well as nanoparticle-based systems widely is important, as we face new challenges posed by multi-drug resistance, expensive diagnostic tools and antimicrobial resistance challenges inspiring the new nanotechnology-based diagnostic and therapeutic tools. This becomes very important in nanomedicine, a field offering a wide possibility of applications from drug-delivery, intracellular sensing to applications in novel medicines and therapeutic approaches using nanomaterials. 
Challenges in reproducibility are not limited to biological experiments with nanoparticles; they also arise from experimental design and technical execution.
Routine particle characterisation and the choice of methods for physico-chemical analysis play key roles in describing systems designed for biomedical applications.
One major challenge is the reproducibility of particle synthesis methods, and its scaleup. Different parameters such as temperature, pH, stirring rate, experimental conditions, impurities, and the choice of glassware during synthesis and equipment can influence particle batches and ultimately particle size, shape, monodispersity, and stability in biological media which all impacts the bio-nano interactions with the living cells. Addressing batch-to-batch reproducibility with robust characterisation methods is essential for planning any serious biological application of nanomaterials, ensuring reliable and reproducible outcomes. 
To mitigate these challenges, a quality scale-up of synthesis is proposed to avoid batch-to-batch variability during synthesis replication. Followed with a well-planned and customised selection of physico-chemical characterisation methods for particle size determination and stability evaluation in relevant media is recommended, in function of the particle size and surface chemistry. Coupling these methods with statistical analysis, though not always customary in nanoparticle analysis, will provide a more robust approach, but also add more complexity over more traditional approaches.
The proposed work aims to answer important questions about endosomal escape, a previously raised issue [1], or at least to contribute understanding. The new proposed method of optical microscopy herein is expected to provide additional insights, such as distinguishing between nanoparticles that have escaped the endosome and those that have directly crossed the membrane into the cytosol, which is not unequivocally possible with using just the traditionally used TEM. 
In bionanotechnology, the use of controls is not a well-established practice. Including controls for particles designed to circumvent endocytosis is proposed as a positive control setup in the studied cellular system. This work will significantly contribute to re-evaluating the use of statistics in nanoscience, as many commonly used biological assays are inadequate when nanoparticle-based drugs are employed. 
Interestingly, the authors report false positives in the selection of articles for evaluation. Including these articles for full justification of the article choice selection would be beneficial. The authors also propose cytotoxicity measurements using more than one commonly used biological assay, demonstrating a robust experimental plan with a clear rationale for using the more sensitive Alamar Blue assay. To correlate results across different assays, the use of FACS is proposed to validate viability results, understand cell death pathways, dynamics, and enhance experimental reproducibility, generating high-quality data with established protocols. [2]
In addition to the planned characterization methods, it is proposed to include Differential Centrifugal Sedimentation (DCS) for high-resolution nanoparticle analysis in relevant biological fluids used for in vitroanalysis. This will assess the overall stability of the dispersions and exclude the formation of aggregates that can significantly impact biological assessment outcomes for ultra-small nanoparticles. [3] Nanoparticle Tracking Analysis (NTA) is also suggested for reproducible nanoparticle concentration measurements, which are important for dosing of the particles in biological experiments.
This study holds a high hope to advance our understanding of the reproducibility in field of nanomedicine by addressing challenges in adapting synthesis to reduce batch-to-batch variability, validating physico-chemical characterization tools, and ensuring the reliability of cell viability tests. It will identify best practices in operative procedures, create opportunities for standardizing measurements and in vitro assessment approaches, and ultimately guide new commercial opportunities, regulatory requirements, and wider applications in futire clinical trials.

References: 

1. Željka Krpetić, Samia Saleemi, Ian A. Prior, Violaine Sée, Rumana Qureshi, and Mathias Brust. Negotiation of Intracellular Membrane Barriers by TAT-Modified Gold Nanoparticles. ACS Nano 2011, 5 (6), 5195-5520. DOI: 10.1021/nn201369k
 
2. Anna Salvati, Inge Nelissen, Andrea Haase, Christoffer Åberg, Sergio Moya, An Jacobs, Fatima Alnasser, Tony Bewersdorff, Sarah Deville, Andreas Luch, Kenneth A. Dawson. Quantitative measurement of nanoparticle uptake by flow cytometry illustrated by an interlaboratory comparison of the uptake of labelled polystyrene nanoparticles, NanoImpact, 2018, 9, 42-50, https://doi.org/10.1016/j.impact.2017.10.004.
 
3. André Perez-Potti, Hender Lopez, Beatriz Pelaz, Abuelmagd Abdelmonem, Mahmoud G. Soliman, Ingmar Schoen, Philip M. Kelly, Kenneth A. Dawson, Wolfgang J. Parak, Zeljka Krpetic & Marco P. Monopoli  In depth characterisation of the biomolecular coronas of polymer coated inorganic nanoparticles with differential centrifugal sedimentation. Sci. Rep. 2021, 11, 6443. https://doi.org/10.1038/s41598-021-84029-8