Over the past decade there has been an exponential increase in the number of research papers utlising nanoparticles for biological applications such as intracellular sensing [1, 2], theranostics [3-5] and more recently drug delivery and precision medicine [6, 7]. Despite the success stories, there is a disconnect regarding current dogma on issues such as nanoparticle uptake and trafficking, nanoparticle delivery via the enhanced permeability and retention (EPR) effect, and endosomal escape. Critical re-evaluation of these concepts both conceptually and experimentally is needed for continued advancement in the field.

 

For this preregistration, Said et al. (2024) focus on nanoparticle intracellular trafficking, specifically endosomal escape [8]. The current consensus in the literature is that nanoparticles enter cells via endocytosis [9, 10] but reportedly just 1-2% of nanoparticles/ nanoparticle probes escape endosomes and enter the cytoplasm [11-13]. There is therefore an apparent paradox over how sensing nanoparticles can detect their intended targets in the cytoplasm if they are trapped within the cell endosomes. To address this fundamental issue of nanoparticle endosomal escape, Lévy and coworkers are carrying out replication studies to thoroughly and transparently replicate the most influential papers in the field of nanoparticle sensing. The aim of these replication studies is twofold: to establish a robust methodology to study endosomal escape of nanoparticles, and to encourage discussions, transparency and a step-change in the field.  

 

 

For this replication study, the authors classified papers on the topic of nanoparticle sensing and subsequently ranked them by number of citations.  Based on this evaluation they selected a paper by Zhu and colleagues [14] entitled “Carbon-Dot-Based Dual-Emission Nanohybrid Produces a Ratiometric Fluorescent Sensor for In Vivo Imaging of Cellular Copper Ions” for their seminal replication study.  To determine the reproducibility of the results from Zhu et al., the authors aim to establish the proportion of endosomal escape of the nanoparticles, and to examine the data in a biological context relevant to the application of the probe.

 

 

The authors plan to replicate the exact conditions reported in the materials and methods section of the selected paper such as nanoparticle probe synthesis of CdSe@C-TPEA nanoparticles, assessment of particle size, stability and reactivity and effect on cells (TEM, pH experiments, fluorescent responsivity to metal ions and cell viability). In addition, Said et al., plan to include further experimental characterisation to complement the existing study by Zhu and colleagues. They will incorporate additional controls and methodology to determine the intracellular location of nanoparticle probes in cells including: quantifying excess fluorescence in the culture medium, live cell imaging analysis, immunofluorescence with endosomal and lysosomal markers, and electron microscopy of cell sections. The authors will also include supplementary viability studies to assess the impact of the nanoparticles on HeLa cells as well as an additional biologically relevant cell line (for use in conjunction with the HeLa cells as per the original paper).

 

The Stage 1 manuscript underwent two rounds of thorough in-depth review. After considering the detailed responses to the reviewers' comments, the recommenders  determined that the manuscript met the Stage 1 criteria and awarded in-principle acceptance (IPA).  

 

The authors have thoughtfully considered their experimental approach to the replication study, whilst acknowledging any potential limitations. Given that conducting such a replication study is novel in the field of Nanotechnology and there is currently no ‘gold standard’ approach in doing so, the authors have showed thoughtful regard of statistical analysis and unbiased methodology where possible.

 

Based on current information, this study is the first use of preregistration via Peer Community in Registered Reports and the first formalised replication study in Nanotechnology for Biosciences. The outcomes of this of this study will be significant both scientifically and in the wider context in discussion of the scientific method.

 

 
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:

 

 

1. Howes, P. D., Chandrawati, R., & Stevens, M. M. (2014). Colloidal nanoparticles as advanced biological sensors. Science, 346(6205), 1247390. https://doi.org/10.1126/science.1247390

 

2. Liu, C. G., Han, Y. H., Kankala, R. K., Wang, S. B., & Chen, A. Z. (2020). Subcellular performance of nanoparticles in cancer therapy. International Journal of Nanomedicine, 675-704. https://doi.org/10.2147/IJN.S226186

 

3. Tang, W., Fan, W., Lau, J., Deng, L., Shen, Z., & Chen, X. (2019). Emerging blood–brain-barrier-crossing nanotechnology for brain cancer theranostics. Chemical Society Reviews, 48(11), 2967-3014. https://doi.org/10.1039/C8CS00805A

 

4. Yoon, Y. I., Pang, X., Jung, S., Zhang, G., Kong, M., Liu, G., & Chen, X. (2018). Smart gold nanoparticle-stabilized ultrasound microbubbles as cancer theranostics. Journal of Materials Chemistry B, 6(20), 3235-3239. https://doi.org/10.1039%2FC8TB00368H

 

5. Lin, H., Chen, Y., & Shi, J. (2018). Nanoparticle-triggered in situ catalytic chemical reactions for tumour-specific therapy. Chemical Society Reviews, 47(6), 1938-1958. https://doi.org/10.1039/C7CS00471K

 

6. Hou, X., Zaks, T., Langer, R., & Dong, Y. (2021). Lipid nanoparticles for mRNA delivery. Nature Reviews Materials, 6(12), 1078-1094. https://doi.org/10.1038/s41578-021-00358-0

 

7. Mitchell, M. J., Billingsley, M. M., Haley, R. M., Wechsler, M. E., Peppas, N. A., & Langer, R. (2021). Engineering precision nanoparticles for drug delivery. Nature Reviews Drug Discovery, 20(2), 101-124. https://doi.org/10.1038/s41573-020-0090-8

 

8. Said, M., Gharib, M., Zrig, S., & Lévy, R. (2024). Replication of “Carbon-Dot-Based Dual-Emission Nanohybrid Produces a Ratiometric Fluorescent Sensor for In Vivo Imaging of Cellular Copper Ions”. In principle acceptance of Version 3 by Peer Community in Registered Reports. https://osf.io/qbxpf

 

9. Behzadi, S., Serpooshan, V., Tao, W., Hamaly, M. A., Alkawareek, M. Y., Dreaden, E. C., ... & Mahmoudi, M. (2017). Cellular uptake of nanoparticles: Journey inside the cell. Chemical Society Reviews, 46(14), 4218-4244. https://doi.org/10.1039/C6CS00636A

 

10. de Almeida, M. S., Susnik, E., Drasler, B., Taladriz-Blanco, P., Petri-Fink, A., & Rothen-Rutishauser, B. (2021). Understanding nanoparticle endocytosis to improve targeting strategies in nanomedicine. Chemical society reviews, 50(9), 5397-5434. https://doi.org/10.1039/D0CS01127D

 

11. Smith, S. A., Selby, L. I., Johnston, A. P., & Such, G. K. (2018). The endosomal escape of nanoparticles: toward more efficient cellular delivery. Bioconjugate Chemistry, 30(2), 263-272. http://dx.doi.org/10.1021/acs.bioconjchem.8b00732

 

12. Cupic, K. I., Rennick, J. J., Johnston, A. P., & Such, G. K. (2019). Controlling endosomal escape using nanoparticle composition: current progress and future perspectives. Nanomedicine, 14(2), 215-223. https://doi.org/10.2217/nnm-2018-0326

 

13. Wang, Y., & Huang, L. (2013). A window onto siRNA delivery. Nature Biotechnology, 31(7), 611-612. https://doi.org/10.1038/nbt.2634

 

14. Zhu, A., Qu, Q., Shao, X., Kong, B., & Tian, Y. (2012). Carbon-dot-based dual-emission nanohybrid produces a ratiometric fluorescent sensor for in vivo imaging of cellular copper ions. Angewandte Chemie (International ed. in English), 51(29), 7185-7189. https://doi.org/10.1002/anie.201109089