Synthesis and study of cell-penetrating peptide-modified gold nanoparticles
Authors: Run Chang
Sandra M Navarro Gallón
Félix Boussoufi
Thomas Jay Webster
Journal: International journal of nanomedicine (2018)
Discipline: PHARMACOLOGY & TOXICOLOGY
Keywords: cell-penetrating peptide; cysteine; fibroblast; gold nanoparticles.
Affiliations: Department of High Performance Materials, Sigma Clermont, Clermont-Ferrand, France., Biotechnology Group, Faculty of Exact and Natural Sciences, Universidad de Antioquia, Medellín, Colombia., Department of Chemical Engineering, Northeastern University, Boston, MA, USA, .

Abstract:  Background: In nanomedicine, gold nanoparticles (AuNPs) have demonstrated versatile therapeutic efficiencies and, in particular, have been developed for the treatment of various cancers due to their high selectivity in killing cancer, not healthy, cells. Methods: In this study, AuNPs were conjugated with the cell-penetrating peptide Cys-(Arg)8-Asp-Ser (CRRRRRRRRGDS) by direct cross-linking of the cysteine's thiol group to the gold surface and a fibronectin-derived RGD group was also used due to its efficacy toward cancer cell targeting and possible promotion of healthy fibroblast functions. Results: Ultraviolet-visible absorbance spectrum and transmission electron microscope images of the synthesized peptide-capped AuNPs (PEP-AuNPs) validated the formation of AuNP aggregates. The presence of peptides on AuNPs was confirmed by Fourier transform infrared spectroscopy and quantified by a bicinchoninic acid assay. After being modified with the arginine-rich peptide, the AuNPs possessed a positive charge, as their zeta potential increased from -23.81±8.43 mV to 8 mV on average. In this manner, an easy method to conjugate AuNPs was shown here. Further, MTS assays were performed using healthy human dermal fibroblasts. After 24 hours of treatment with PEP-AuNPs, the cell density increased dramatically to around 25,000 cells/cm2. Results further showed a very high half-maximal inhibitory concentration of 69.2 µM for the PEP-AuNPs (indicating low toxicity). Conclusion: The results showed for the first time the ability of PEP-AuNPs to promote human dermal fibroblast cell viability, which after further investigation, may show an ability to replace cancerous tissue with healthy soft tissue. 

Retracted: No

PubPeer comments:
 **Figure 2A**, images of immunofluorescence results for anti-CD51 (red) and CD68 (green) staining. An obvious image overlap was observed between the upper panel and the lower panel (**marked in red box**). In nature, the authors have used the different view fields from the same section staining. ![file] ![file] ![file] , 
 Correspondingly, Figure 2A, the **DAPI staining** also showed the problem of image uses (**marked in yellow box**) as the **HE staining** above. They all come from the same section staining resource. ![file] ![file] ![file] , 
 Thanks for your comments. After checking the original data, we found that the two sets of pictures in Fig 2A came from the serial sections of the same patient. Because the expression of CD68 and CD51 is more significant, we chose these two sets of images as representative images in the main text, and our manuscript did not mention that these two sets of representative images come from different patients. At the same time, we provide more patient data in supplementary figure S3B to support the co-expression of CD68 and CD51 in pancreatic cancer.We will contact with journal editor to make a correction. This correction has not changed the interpretation or the original conclusions of this work. Best wishes. , 
 #3 First of all, since the author team has admitted that the two partially repeated images are from the same slice, why not shoot these representative areas directly in a larger field of view? Second, the author s team acknowledges that these representative images are the same slice from the same patient. Is it true that only the slice of this patient is representative, and there is no such obvious co-expression in other patients? Third, since the author s team believes that the co-expression of CD51 and CD68 is common, it should capture more tissue sections of different patients for observation and research, rather than blindly emphasizing that these repeated pictures come from the same patient. Such an approach is just ride off on side issues. I hope to see representative research results **from different patient sections**. , 
 Carefully observe the sphere formation assays of **SW1990 in Fig. 2F**. The shooting background of the cells in the control group is exactly the same as that in the co-culture of M1, and the **red dotted box** is an obvious representative repeated background area. It can be determined that the two pictures of sphere formation assays were taken from the same group of cells, rather than the cell spheroidization experiment under two different group cultures. The second so-called cell mass co-cultured with M1 is actually the first picture of the results of the cell mass as the control group cultured **at different time points**. ![file] ![file] **The shooting background of the two cell clusters is completely consistent (the scattered round cells are non-clustered single cells, and their size, shape and distribution position are completely consistent), indicating that the two pictures are the same cell cluster photographed at different time points**. **The cell clusters under the condition of co-culture with M1 should be photographed at the time point when the culture time is longer, and the cell clusters are larger, that is, after the culture time is extended**. , 
 #3 Figure 2G, Figure 4C: Similarly, the authors team is suggested that when showing the co-expression of CD51 and CD68 on tissue sections, more different patients are needed. In addition, in animal experiments, the authors should also show the picture results of tumor growth of **all mice** (the author s team mentioned in the research method that mice are randomly grouped, but did not specify the specific number), rather than just a picture of **one mouse**, and only **one tumor sample** of one mouse in each group. This so-called only one representative mouse specimen instead of rigorous experimental conclusions is not common, and is obviously unreliable. ![file] ![file] **** , 
 #3 Data problem could be found in **Table 1** In the statistical results of the **Age group**, the number of patients older than 60 and patients younger than 60 is almost the same in the high and low expression of CD51, and there should be no statistical difference in my first feeling. Furthermore, when recalculated the P value by myself, based on the Pearson Chi-square test method used by the authors, the recalculated P value is **0.2430 (chi-square = 1.363)**. ![file] This could be confirmed in the **Table 2**, a non-significant difference was found in the Age column in the uni-variate analysis. ![file] , 
 #3 Also in Table 1, unexplainable data in **lymph node status** was revealed. All the variates of **AJCC stage, tumor stage and lymph node stage** were associated with significant difference based on the expression levels of CD51, however, when looked into the specific number carefully, in the **Lymph node stage**, the number of N0 node was much more reported than the number of N1 node, which was obviously acceptable and illogical. As the authors pointed out that **CD51 was positively associated with the poor prognosis of PDAC patients, in other words, CD51 expression was correlated with the higher TNM stage in theory**. Thus, the obviously smaller number in N1 lymph node compared with the N0 was problematic. Kindly requested the authors discuss it, thank you. ![file] , 
 #3 The authors stated in the Results Section, that  These results were further confirmed by analyses of the **TCGA database**, collectively supporting the oncogenic role of CD51 (Fig. 1G-I) . ![file] Of note was the observation that in the survival analysis from the TCGA database, there existed a **non-significant difference (P = 0.088)** between the high and low expression of **CD51 (ITGAV)** in PAAD, actually. ![file] See: http: gepia.cancer-pku.cn detail.php?gene=CD51 The authors did not mark the P value in their paper. , 
 #3 **Figure 6A**, the expression of macrophage-secreted cytokines was assessed by qRT-PCR. The results showed that CD51 knockdown decreased all the levels of these cytokines. ![file] However, TNF-u03b1 secretion is one of the main characteristics of M1-macrophage. Previously, the authors have clearly demonstrated that CD51 drives macrophage polarization toward an M2-like phenotype. In Figure 6A, when CD51 was knocked down already, the mark of M1-like, TNF-u03b1 should be higher rather than become lower. , 
 #3 **Figure 6G**, the protein expression of CSC-related transcription factors was assessed. It feels non-sense, the design of the WB experiment was purposeless actually, in the red box. There is no need to testify that TGF-u03b21 could upload the expression of these CSC-related genes in tumor cells, which have been already confirmed many times in the previously published literature. ![file] It is suggested adding the **shCD51#1 CM + TGF-u03b21** and **shCD51#2 CM + TGF-u03b21** groups in the WB experiment, just as those in Figure 6E and Figure 6F, to better elucidate the mechanisms. ,