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Replicating, Revising and Reforming: Unpicking the Apparent Nanoparticle Endosomal Escape Paradox
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Replication of “Carbon-Dot-Based Dual-Emission Nanohybrid Produces a Ratiometric Fluorescent Sensor for In Vivo Imaging of Cellular Copper Ions”
Abstract
Recommendation: posted 21 July 2024, validated 22 July 2024
Linnane, E. and Yamada, Y. (2024) Replicating, Revising and Reforming: Unpicking the Apparent Nanoparticle Endosomal Escape Paradox. Peer Community in Registered Reports, . https://rr.peercommunityin.org/articles/rec?id=610
Recommendation
Level of bias control achieved: Level 6. No part of the data or evidence that will be used to answer the research question yet exists and no part will be generated until after IPA.
List of eligible PCI RR-friendly Journals:
- In&Vertebrates
- Peer Community Journal
- PeerJ
- PeerJ Materials Science
- Peer J Organic Chemistry
- Royal Society Open Science
The recommender in charge of the evaluation of the article and the reviewers declared that they have no conflict of interest (as defined in the code of conduct of PCI) with the authors or with the content of the article.
Evaluation round #2
DOI or URL of the report: https://osf.io/kf9qe/
Version of the report: 1
Author's Reply, 26 Jun 2024
Decision by Emily Linnane and Yuki Yamada
, posted 12 Jun 2024, validated 12 Jun 2024
The authors have provided an overview of the research question/rationale and three key aims for this replication study, as well as an in-depth outline of methods and protocols both in line with the original study and also with additional controls where necessary.
Given the challenge presented in the field, this study, when published, will have a significant impact in the field.
The manuscript has been reviewed and there are a few minor comments below to address before proceeding to recommendation. Once these minor comments and suggestions have been addressed by the authors we can proceed with the recommendation.
Reviewed by Cecilia Menard-Moyon
, 24 Apr 2024
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.
Reviewed by Zeljka Krpetic
, 11 Jun 2024
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
Evaluation round #1
DOI or URL of the report: https://osf.io/kf9qe/
Version of the report: 1
Author's Reply, 25 Feb 2024
Decision by Emily Linnane and Yuki Yamada
, posted 14 Dec 2023, validated 15 Dec 2023
I am a co-recommender of this manuscript with Emily. First of all, we are very pleased to see your submission from this new research field! I have seen a variety of manuscripts in the PCI RR, and here I would like to comment on to help you proceed smoothly toward the peer review process.
I encourage you to take another closer look at the Guide for Authors for more information about Registered Reports and the PCI RR system. The “criteria" in 2. Submission requirements are particularly important.
As you may already know, to proceed with peer review in this system, a solid background for the hypothesis is necessary, and it must be clear in advance which experimental results will be required to support the hypothesis. Your protocol appears to present multiple hypotheses. The PCI RR provides a study design template that summarizes each hypothesis. Your table is somewhat different from this, but I think it would be relatively easy to modify it into our study design template. I believe it should at least be revised before undergoing peer review for this submission. Whenever possible, what is written here should also be explained in the text. Then, after revising the study design template and related materials, have Emily scrutinize them for content.
Looking forward to seeing the revised manuscript again.
Yuki Yamada
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I am very pleased to be a co-recommender on this interesting replication study. I have below some suggestions for revision before sending to review.
In keeping with the submission criteria and as mentioned by Yuki, the full study design table needs to be included. In addition to this, the structure of the article needs to be similar to that of a final article and therefore some amendments to the manuscript structure would be useful at this stage to fit with this format.
The authors have provided an overview of the research question/rationale and three key aims for this replication study, as well as an in-depth outline of methods and protocols both in line with the original study and also with additional controls where necessary.
I also include below some other suggestions for revision before submission to peer review:
With reference to the assessment of materials on cell viability (Assessing cytotoxicity of CdSe@C-TPEA in Hela cells, 1.14.1. MTT cytotoxicity assay and 1.14.2. Alamar Blue assay)
- To measure cytotoxicity of the probes, the authors of the original article used the MTT assay. Given the limitations and pitfalls recently reviewed, in addition to the MTT assay, the authors plan to assess cytotoxicity with the more sensitive Alamar Blue assay. I would suggest it may also be important to include a cytotoxicity assay which isn’t impacted by cellular metabolism as a read out, e.g. SRB assay. Also, it might be valuable to include the HepG2 cells in this experimental section too, as it is not known if these cells would be more sensitive to the nanoparticles and therefore to have a cytotoxicity readout would be usefulas these are including in the sensing experiments.
- Can the authors comment on the number of repeats to be carried out and statistical analysis
The authors note that typically ~1-2% of nanoparticles are reported to escape. They plan to test whether the localization of the probes is in line with this expectation or whether instead a high proportion of nanoparticles has escaped endosomes, by studying intracellular localization through several different microscopy experiments.
Regarding the plans for fixed cell immunofluorescence with endosomal and lysosomal markers (1.15.2. Immunofluorescence Imaging with CdSe@C-TPEA in HeLa cells using markers (EEA1, LAMP-1, LysoTracker®)
- How will the percentage of nanoparticles (escaped or in endosomes/ lysosomes) be calculated? The cells will be incubated for 2 hours, how will the total amount of particle internalised be assessed (e.g. what about the material remaining in the culture media?)
- What analysis will be carried out/what statistics will be used?
- How many experimental repeats will be carried out?
- If co-localisation with endosomes/ lysosomes will be assessed, what software will be used (e.g. Image J or commercial software). Will any correlation analysis be carried out?
- The authors state that at least 30 cells will be used per experiment,is this enough cells? how will bias be removed from the analysis in the selection of cells (e.g. will this be automated / or done blind?)
Regarding the plans for live cell fluorescence Imaging with ( 1.15.3. CdSe@C-TPEA in HeLa cells using marker CellLight™ Early Endosomes-RFP BacMam 2.0, CellLight™ Late Endosomes-RFP BacMam 2.0, LysoTracker®)
- What analysis will be carried out on the Incucyte®?
- How will this data be quantified/ represented?
- How many repeats will be carried out?
The experimental plans: 1.17. Intracellular Cu quantification in Hela and HepG2/C3a cell lines using inductively coupled plasma mass spectrometry (ICP-MS)
- Can the authors comment on the quantification by ICP-MS, how the data will be analysed and number of conditions and repeats etc?
Minor experimental suggestions and comments (if applicable but not necessary for replication study)
- The authors from the original paper (Zhu et al) incubate HeLa cells with the probe and phorbol-12,13-dibutyrate PDBu - a compound they state is known to increase the endocytic activity. In the replication study this PDBu is therefore also included in the experimental plans. Is there any rationale for including another set of experimental conditions without the PDBu? This may be beyond the scope of the replication study and is not necessary for assessment of the aims outlined in the paper introduction, however inclusion may impact the overall conclusion on endosomal escape of the particles if endocytosis is affected by this compound. Also, does PDBu have comparable impact on endocytic activity in HepG2 cells as HeLa cells?
- Would also recommend STR profiling of cells at least once during the study as well as mycoplasma testing
- If technically possible (I don’t know if this would be and it is likely to be beyond the scope of this study) but including a non-microscopy-based quantification of probes would be useful, e.g. performing cellular fractionation to separate membrane bound vesicles from the cytoplasm then carrying out e.g mass spec (if possible) to provide a more robust quantitative measure of probe present
I also look forward to seeing the revised manuscript again.
Emily Linnane