Near infrared fluorescent peptide nanoparticles for enhancing esophageal cancer therapeutic efficacy

Various types of nanoparticles have been proposed for targeted drug delivering, imaging, and tracking of therapeutic agents. However, highly biocompatible nanoparticles with structure-induced fluorescence and capability to conjugate with biomarkers and drugs remain lacking. This research proposes and synthesizes fluorescent nanoparticles (f-PNPs) assembled by cyclic peptides to combine imaging and drug delivering for esophageal cancer (EC). To achieve tumor targeting, f-PNPs are first conjugated with RGD moieties to selectively target EC cells via αvβ3 integrin; the nanoparticles are then embedded with epirubicin (EPI). Cell viability assays and analysis of tissue histology reveal that EPI-loaded RGD-f-PNPs (RGD-f-PNPs/EPI) led to significantly reduced cardiotoxicity and improved anti-tumor activity compared to EPI alone. Moreover, the drug delivery to tumor sites and therapeutic responses could be monitored with near-infrared fluorescence using RGD-f-PNPs/EPI. This unique nanoparticle system may lead to potential approaches for bioorganic fluorescence-based delivering, imaging, and drug release tracking.

1. Since the cell specificity depends on the recognition of alpha(v)beta(3) integrin by RGD, it would be nice to do a simple knockdown experiment on KYSE30 or 270 cells to show that the specificity of RGD-f-PNPs depends on the presence of integrin. 2. Epirubicin is a chemotherapeutic agent commonly used for esophageal adenocarcinoma or gastroesophageal junction adenocarcinoma. Testing this compound for esophageal squamous cell carcinoma cells (KYSE cells) needs to be justified. 3. The doses of 1.5mg/kg RGD-f-PNPs/EPI and 6mg/kg EPI need to justified. Mouse esophagus may servce as control of toxicity to the normal esophageal epithelial cells. Alfalfa free diet is needed to eliminate autofluoresecence in the stomach area. 4. This manuscript contains quite some grammatical and spelling errors. English editing is a must. 5. In Introduction, it is not correct that "currently EC diagnosis is primarily based on individual surgeon's personal experince to interpret patients' signs of symptoms ..." It will surely help to let a clinician with esophageal cancer experience go through this section and highlight the potential translational value.
Reviewer #2: Remarks to the Author: This manuscript reports on nanoparticles made of cyclic peptide that, together with RGD and Zinc ions, can generate both fluorescence and directly target EC cells for chemo-drugs delivery and improve antitumor activity. This manuscript is most likely a continuation to the group's former paper: "Bioinspired fluorescent dipeptide nanoparticles for targeted cancer cell imaging and real-time monitoring of drug release". I would suggest to refer the following comments: (1) Usually, intrinsic fluorescence is considered fluorescence of the material itself, without any guest material supplement. See for example the paper:"Reconstructive Phase Transition in Ultrashort Peptide Nanostructures and Induced Visible Photoluminescence". In this manuscript, both RGD and Zinc ions generates the fluorescence and NOT the peptide material itself. Thus, I think that the word "intrinsic" is misleading.
(2) There are several reposrts on fluorescent peptide-based materials that are relevant to this manuscript, for example "Peptide-C onjugated Fluorescent Silicon Nanoparticles Enabling Simultaneous Tracking and Specific Destruction of C ancer C ells". The authors should explain the difference between the development to this paper.
(3) The role of the Zinc ions is not clear -if the RGD produce the fluorescence, why do you need the Zinc ions?
To summarize, I recommand to AC C EPT the paper to Nat. C omm., after referring to these comments.

Manuscript NCOMMS-17-29944
We appreciate both reviewers' recognition regarding the significance and potential translational value of this study. We also thank them for their critical review and constructive comments on the manuscript, which have helped us to designed more rigorous experiments and reshaped the manuscript. The current version has incorporated some new data on esophageal adenocarcinoma cells, in vitro and in vivo studies of esophageal cancer cell lines with knockout of integrin alpha-V gene, and toxicity analysis of mice esophageal epithelial. The detailed point-by-point responses are enclosed below. With these new data and substantial changes in the manuscript according to the reviewers' suggestions, we hope this revised version of our manuscript can be published in Nature Communication.

Responses to Reviewer #1:
"This manuscript by Fan et al report an exciting delivery system for treating esophageal cancer. RGD-f-PNPs were designed to deliver chemotherapeutic agents to esophageal cancer through the interaction between RGD and alpha(v)beta (3) integrin. Epirubicin was tested as an example of such chemotherapeutic agents. Both in vitro and in vivo data suggested specificity and anti-cancer efficacy of the delivery system. Overall this study is quite significant in its potential translational value. However, this reviewer as a biologist can only comment on the biology of this manuscript due to lack of chemical/engineering expertise." Respond: We appreciate your encouraging comments.
1. Since the cell specificity depends on the recognition of alpha(v)beta(3) integrin by RGD, it would be nice to do a simple knockdown experiment on KYSE30 or 270 cells to show that the specificity of RGDf-PNPs depends on the presence of integrin.
Respond: Thank you for the suggestion. We have performed CRISPA/Cas9 based gene knockdown assay and the new data are presented in new Supplementary Fig. 7. The effect of knockout of alpha(v)beta(3) integrin (also called CD51) was confirmed by immunofluorescent microscopy imaging. The cells lacking CD51 showed no plasma membrane fluorescent signal from the RGD-f-PNPs. But since CD51 is essential for tumor formation and KYSE-30 cells lacking CD51 couldn't form tumor, we were not able to compare the RGD-f-PNP uptake in xenograft mice injected with KYSE-30/CD KO cells.
2. Epirubicin is a chemotherapeutic agent commonly used for esophageal adenocarcinoma or gastroesophageal junction adenocarcinoma. Testing this compound for esophageal squamous cell carcinoma cells (KYSE cells) needs to be justified.
Respond: We agree. Epirubicin can effectively induce cell death in many cancer cells including esophageal squamous cell carcinoma. Although it has been used often for patients with esophageal adenocarcinoma and gastroesophageal junction adenocarcinoma, more clinical trials demonstrated their benefits for ESCC patients as well (Andreyev et al, Eur J Cancer. 1995;Honda et al, Dis Esophagus. 2010;Kosugi, et al Scand J Gastroenterol. 2005). We included a set of data on esophageal adenocarcinoma cell line OE33. As shown in Supplementary Fig. 7, the esophageal adenocarcinoma cells (OE33) have higher expression of alpha(v)beta(3) integrin, and RGD-f-PNPs preferentially bind to these cells. Taking together, our data suggest that the RGD-f-PNPs/EPI is suitable for both esophageal squamous cell carcinoma and adenocarcinoma. Thus, this study provides evidence to show that EPI embedded RGD-f-PNPs could be a chemotherapeutic agent for ESCC. We included these points into this revised manuscript.