The integrin facilitated internalization of fibronectin-functionalized camptothecin-loaded DNA-nanofibers for high-efficiency anticancer effects
Authors: Wing-Fu Lai
Ammara Saleem
Ghulam Jilany Khan
Muhammad Naveed
Jahanzeb Mudassir
Abdoh Taleb
Muhammad Furqan Akhtar
Muhammad Tayyab Ansari
Farhan Siddique
Reyaj Mikrani
Mirza Muhammad Faran Ashraf Baig
Saba Ashraf
Journal: Drug Delivery and Translational Research (2020)
Discipline: PHARMACOLOGY & TOXICOLOGY
Keywords: A549 cells; Camptothecin (CMPT); DNA-nanofibers (DNA-NFs); Fibronectin (FN); Integrin receptors.
Affiliations: State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, 210023, People's Republic of China. ., Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Bahauddin Zakariya University, Multan, 60000, Pakistan. ., Department of Applied Biology and Chemical Technology, Hong Kong Polytechnic University, Hong Kong Special Administrative Region, People's Republic of China., School of Life and Health Sciences, The Chinese University of Hong Kong, Shenzhen, People's Republic of China., Nishtar Medical University and Hospital, Multan, 60000, Pakistan., Department of Pharmacology, Faculty of Pharmaceutical Sciences, Government College University Faisalabad, Faisalabad, Pakistan., Riphah Institute of Pharmaceutical Sciences, Riphah International University, Lahore, Pakistan., School of Basic Medicine, and Clinical Pharmacy, China Pharmaceutical University, Nanjing, 211198, Jiangsu, People's Republic of China., School of Pharmacy, Nanjing Medical University, Nanjing, China., Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Bahauddin Zakariya University, Multan, 60000, Pakistan., Faculty of Pharmacy, University of Central Punjab, Lahore, 54570, Pakistan.

Abstract:  Camptothecin (CMPT) in a free form is extremely cytotoxic as well as hydrophobic drug, and is considered to be highly contagious for systemic administration. The fibronectin (FN)-functionalized DNA-based nanocarrier has been designed to load CMPT and target integrin (αvβ3) receptors which are highly expressed on the A549 cancer cells. Here, we report DNA nanocarrier in the form of DNA-nanofibers (DNA-NFs) capable of loading CMPT via strand intercalation in the GC (base pairs)-rich regions of the DNA duplex. Hence, our keen purpose was to explore the potential of DNA-NFs to load CMPT and assess the improvements of the outcomes in terms of enhanced therapeutic effects to integrin-rich A549 cancer cells with reduced cytotoxic effects to integrin-lacking HEK293 cells. DNA-NFs were formulated as a polymer of DNA triangles. DNA triangles arranged in a programmed way through the complementary overhangs present at the vertices. DNA triangles were primarily obtained through the annealing of the freshly circularized scaffold strands with the three distinct staple strands of specific sequences. The polymerized triangular tiles instead of forming two-dimensional nanosheets underwent self-coiling to give rise to DNA-NF-shaped structures. Flow cytometry and MTT assays were performed to observe cytotoxic and apoptotic effects on integrin-rich A549 cancer cells compared with the integrin-deficient HEK293 cells. AFM, native-page, and confocal experiments confirmed the polymerization of DNA triangles and the morphology of the resulting nanostructures. AFM and confocal images revealed the length of DNA-NFs to be 3-6 μm and the width from 70 to 110 nm. CMPT loading (via strands intercalation) in GC-rich regions of DNA-NFs and the FN functionalization (TAMRA tagged; red fluorescence) via amide chemistry using amino-modified strands of DNA-NFs were confirmed through the UV-shift analysis (&gt; 10 nm shift) and confocal imaging. Blank DNA-NFs were found to be highly biocompatible in 2-640 μM concentrations. MTT assay and flow cytometry experiments revealed that CMPT-loaded DNA-NFs showed a dose-dependent decrease in the cell viability to integrin-rich A549 cancer cells compared with the integrin-deficient HEK293 cells. Conclusively, FN-functionalized, CMPT-loaded DNA-NFs effectively destroyed integrin-rich A549 cancer cells in a targeted manner compared with integrin-deficient HEK293 cells. Grapical abstract. 

Retracted: No

PubPeer comments:
 The images in Figure 4 of this paper appear to also overlap with Figure 4 of another paper by the same first author, i.e. Mirza Muhammad Faran Ashraf Baig and Xing-Hua Xia, Applied Nanoscience (2020), DOI: 10.1007 s13204-020-01293-2 - https: pubpeer.com publications 1019754110D74381242E47B7FA5572 Can the authors please clarify if the NWs (nanowires) from this paper are the same as the NF (nanofibers) from the DDTR paper? And can they also clarify if these images might all be showing the same specimen? ![file] , 
 Some further investigation showed that Applied Nanoscience (2020), DOI: 10.1007 s13204-020-01293-2, the DNA nanowires (NW) are based on Carboplatin (CRPT), while in Drug Delivery and Translational Research (2020), DOI: 10.1007 s13346-020-00820-6, the DNA nanofibers (NF) are based on Camptothecin (CMPT). Thus, it looks as if the NWs from one paper are different from the NFs in the other paper, and the figures therefore should not overlap or look similar. Can the authors please clarify if this is correct? Figures 2 for example look very similar. ![file] Figure 3 also have unexpected similarities (green and cyan boxes): ![file] , 
 Got interested to Dr Baig s work after that pubpeer comment: https: pubpeer.com publications E695E673D46779A362E6749254A8D8 Dr Baig published 40 papers in 2020, 13 of them as 1st authors - Seems unreasonably high. Picked a random paper, turned out Dr. Bik already commented on it and I found more alterations on that same figure 4. Note that most part have been copied - resized expanded - pasted. Cyan bar denotes an unusual difference in size within the same panel Sharp cut-paste edge pointed by arrow ![file] , 
 Fig 3a. I have concerns. ![file] , 
 #3 Fig 4b.  Confocal imaging of TAMRA-tagged CMPT-loaded DNA-NF also showed similar morphology . ![file] As well as the similarity of the two upper panels (after a 90-deg rotation and rescaling), an area is the same in the two left-had panels (after a 180-deg flip). --------------- UPDATE: This comment just duplicates comment #1. Apologies. ----------------------  Sharp cut-paste edge pointed by arrow - that could just be an artefact introduced by the rotation and rescaling. , 
 Actually these studies are the part of same project. Shape resembles with nanofiber or nanowire. The material is same. Therefore, characterization overlapped. But application was different. , 
 #1 Here s my take on the visual elements shared among the panels of the present Fig 4 (above) and Fig 4 of [Baig Xia (2020)] (below). ![] Not to forget the duplicated visual elements **within** panels. ![file] , 
 I arranged wrongly. But I have a raw data in previous dates to show that I really performed this work. I will share soon. , 
 This gets complicated, but a third paper might also show some of the same confocal images. A list of all three papers: * Mirza Muhammad Faran Ashraf Baig and Xing-Hua Xia, Applied Nanoscience (2020), DOI: 10.1007 s13204-020-01293-2 - https: pubpeer.com publications 1019754110D74381242E47B7FA5572 - showing Carboplatin (CRPT)-based DNA nanowires * Mirza Muhammad Faran Ashraf Baig et al. Drug Delivery and Translational Research (2020), DOI: 10.1007 s13346-020-00820-6 - https: pubpeer.com publications 01794C3A20849640AEC54C15A8B99E - showing Camptothecin (CMPT)-based DNA nanofibers * Mirza Muhammad Faran Ashraf Baig et al., Journal of Molecular Liquids (2020), DOI: 10.1016 j.molliq.2020.113785 - https: pubpeer.com publications 7BF60F063AAE7C5D021A1318B1FDDC - showing Bleomycin-loaded DNA-nanotubes An illustration: ![file] Looking at the relative position of the yellow, blue, and orange boxes, it appears that these are not just simply overlapping images, but that features might have been rearranged. Can the authors please clarify how these three differently synthesized DNA nanowires nanotubes nanofibers could yield such similar photos? , 
 ![file] exported image of 2017 exported tiff image , 
 Sorry, I had many many AFM images. But I thought all look similar. And after overall functionlization, also look similar. So it was my ignorennce to show the similar morphology again and again with some alterations. But it s reality that nanotubes (or nanowires and nanofiebrs etc) all look like similar. I have provided more AFM images to show. ,