Phase-shift, targeted nanoparticles for ultrasound molecular imaging by low intensity focused ultrasound irradiation
Authors: Maoping Li
Pan Li
Weiyang Zhang
Haitao Ran
Yong Chen
Lan Hao
Jianxin Liu
Zhigang Wang
Yuanyi Zheng
Hua Luo
Dong Wang
Kunyan He
Junchen Chen
Journal: International journal of nanomedicine (2018)
Discipline: PHARMACOLOGY & TOXICOLOGY
Keywords: acoustic contrast agent; folic acid; nanoparticles; phase-shift; targeted.
Affiliations: Department of Ultrasound, The First Affiliated Hospital, Chongqing Medical University, Chongqing 400016, China., Institute of Ultrasound Imaging, Chongqing Medical University, Chongqing 400010, China, ; ., Chongqing Protein way Biotechnology Co., Ltd., Chongqing 400039, China., The Fifth Affiliated Hospital of Sun Yat-sen University, Guangzhou, 519000, China., Hunan Key Laboratory of Skin Cancer and Psoriasis, Changsha, 410008, China.

Abstract:  Purpose: Ultrasound (US) molecular imaging provides a non-invasive way to visualize tumor tissues at molecular and cell levels and could improve diagnosis. One problem of using US molecular imaging is microbubbles challenges, including instability, short circulation time, and poor loading capacity and penetrability. It is urgent to design new acoustic contrast agents and new imaging methods to facilitate tumor-targeted imaging. In this study, phase-shift poly lactic-co-glycolic acid (PLGA) nanoparticles modified with folate as an efficient US molecular probe were designed and the long-term targeted imaging was achieved by low-intensity focused US (LIFU) irradiation. Methods: A new 5-step method and purification procedure was carried out to obtain uniform folic acid polyethylene glycol PLGA (PLGA-PEG-FA), the structure of which was confirmed by 1H nuclear magnetic resonance spectroscopy and thin-layer chromatography. Perflenapent (PFP) was wrapped in PLGA-PEG-FA by a double emulsion solvent evaporation method to obtain PFP/PLGA-PEG-FA nanoparticles. The targeted ability of the resulting nanoparticles was tested in vivo and in vitro. LIFU irradiation can irritate nanoparticle phase-shift to enhance tumor imaging both in vivo and in vitro. Results: PLGA-PEG-FA was a light yellow powder with a final purity of at least 98%, the structure of which was confirmed by 1H nuclear magnetic resonance spectroscopy and thin-layer chromatography. Highly dispersed PFP/PLGA-PEG-FA nanoparticles with spherical morphology have an average diameter of 280.9±33.5 nm, PFP load efficiency of 59.4%±7.1%, and shells, thickness of 28±8.63 nm. The nanoparticles can specifically bind to cells expressing high folate receptor both in vivo and in vitro. Ultrasonic imaging was significantly enhanced in vitro and in vivo by LIFU irradiation. The retention time was significantly prolonged in vivo. Conclusion: Phase-shift PFP/PLGA-PEG-FA nanoparticles induced by LIFU can significantly enhance ultrasonic imaging, specifically targeting tumors expressing folate receptor. As a potential targeting acoustic molecular probe, PFP/PLGA-PEG-FA nanoparticles can be used to achieve targeted localization imaging. 

Retracted: No

PubPeer comments:
 Figure 4A: Several particles appear to be visible multiple times. ![file] , 
 Figure 4B. Some unexpected similarities within the same photo. ![file] , 
 Figure 7B shows photos of 8 different dilutions of nanoparticles. The center parts of these photos look all different, but the background of most (but not all) of these photos shows unexpected similarities. ![file] , 
 #3 Here s a shot in the dark explanation for what you noted in 7B. The experiment involves focusing low intensity ultrasound onto solutions of nanoparticles of various concentrations, which causes phase changes and the formation of microbubbles that are imaged. If the solutions are held in a gel mold that remains in a fixed position relative to the ultrasound source and detector, then the areas outside of where the ultrasound is directed would be expected to be very similar. There s not much detail in the methods section, but it seems like a distinct possibility. The same situation occurs when imaging material on a fixed microscope stage; sometimes irregularities like dust, scratches etc appear on images of different specimens that are photographed sequentially. Disclosure: not in the field and not involved with the authors institution. , 
 #1 Thank you for your comments. We just found the access to visit this website. Figure 4A was to show the nanoparticles by the optical microscope. We tried to display their round shape, small size and uniform distribution clearly, so part of the original photo which was nonhomogeneous area was cut. And the number of nanoparticles is extremely huge, they looks so similar under the optical microscope due to the resolution limitation of the optical microscope and apperaed as round dots. We do not need this figure to do any date analyses, the following results and conclusion were not affected. Attachment is one of the original photo. We are sorry for this less rigorous modification.![file] , 
 #5 Perhaps the journal can provide you with guidance on how to address this in the published paper. I do not believe modification of raw data is a generally acceptable practice, especially without explanation, regardless of the purpose. , 
 Dear Dr. Li, It is always great if the authors take away any concern about potential duplications in figures in scientific papers by providing the original, uncropped versions of these figures. So thank you for providing the uncropped photo for Figure 4. Unfortunately, the uncropped photo you provide in comment #5 bears little relation to the published Figure 4. I do not see an area of overlap, but perhaps you can point that out. What is of more concern, the raw, uncropped photo for Figure 4 that you provided in #5 appears to have many repetitive areas. There are many more and much larger repetitive-looking areas in this newly provided photo than in the published Figure 4. Here is your new photo, in which I have highlighted areas that look very similar by boxes of the same color. Could you provide an explanation how little nanoparticles floating around in a solution can end up in such remarkably similar constellations? ![file] , 
 #7 Dear Dr. Elisabeth M Bik: Thank you for your comments. I am the first author in this paper. I feel so sorry and ashamed about the modified pictures due to my ignorance. And I just found I provided the wrong original picture to my corresponding author in hurry during my working time. I just attached the uncropped photo of Figure 4A. Actually, it is totally unnecessary to make these modifications. We only aimed to show the appearance of nanoparticles under the optical microscope, which proved that we successfully prepared them. There is no any data information included in this figure which contributed to publish this paper. I am so sorry for this less rigorous modification. Attachment is the original photo. We will contact the editor office to correct this error. ![file] , 
 Maoping Li, as a learning experience for readers, could you provide some details about how your  original image provided today evolved into Figure 4A in the published paper. I have set both images side-by-side, and I am struggling to identify similar features. Perhaps you could point them out to the readership. https: imgur.com a wXXDWYb , 
 Dear Dr. Li, here is my side-by-side comparison of Figure 4A as published in the paper (left) and the original photo you provided on Feb 28 in comment #8 (right). Unlike the comment in #9 I do see the similar features, and I have marked the new original with a blue rectangular box to show the part that was included in the paper as Figure 4A. I have also marked the original, single-occurrence  bubbles that appeared to be duplicated in the published version. So while I do agree that indeed this new provided original image appears to be nearly identical to published Figure 4A, I am not sure why so many of the  bubbles were duplicated in the published Figure. ![file] Could you provide some insights why you modified the photo? You wrote  Actually, it is totally unnecessary to make these modifications. We only aimed to show the appearance of nanoparticles under the optical microscope, which proved that we successfully prepared them. There is no any data information included in this figure which contributed to publish this paper.  If it was unnecessary, why did you and or your coauthors make these modifications? I also disagree with your statement that there is no data information included in this figure. The photo shows you and your coauthors successfully prepared the nanoparticles, which appears to be a very huge piece of evidence in this paper. , 
 #10 Dear Dr. Elisabeth M Bik: Thank you for your reply. Both the original and modified photos can definitely prove that we had successfully prepared these nanoparticles as you marked with  bubbles . The difference is that I covered up some unnecessary bigger-size particles (mainly shown in right area) and impurities with duplicated nanoparticles in the modified photo, which made it looks clean and tidy. During the process of nanoparticles preparation, it is inevitable to produce some impurities and bigger-size particles which are normal and totally acceptable for the publication. We only aimed to display the general appearance and shapes of these nanoparticles in this picture as we observed under the microscope, no any data information were included. And we then measured the size of these nanoparticles by dynamic light scattering with a Malvern Zetasizer Nano ZS unit (showed in Fig.4D) (Malvern Instruments, Westborough, MA, USA), which also could confirmed the successful preparation in this study. I m sorry I made this modification by myself, as I had very limited experience to publish paper as a clinician. , 
 Astonishingly, the journal has published this correction: August 11, 2020  The authors have advised that they covered up some larger-sized particles and impurities to enhance the aesthetic of the image shown for Figure 4A, on page 3913. The authors also removed some impurities and enhanced certain aspects of the image shown for Figure 4B. These two images were used to display the general appearance and shapes of the nanoparticles the authors had successfully prepared in the study. The editor determined that while the images had been altered it did not affect the conclusions of the study. The authors wish to apologise for these errors. The correct Figure 4 is shown below. ![file] , 
 Despite all the back and forth over this paper, it seems that at least one additional issue remains unaddressed. The images in Figure 6A and Figure 6C seem to overlap. *** ![file] *** Acknowledgments. The authors are grateful to Chao Yu for technical support with flow cytometry and to Donghan Yang for providing helpful suggestions. This work was supported by the National Natural Science Foundation of China (Grant Nos 81227801, 81601513, 81630047, 31630026, 81371578, 81571688, 81371718), National Distinguished Young Scholars (Grant No 81425014), National Research Program of China (973 Program, Grant No 2011CB707905), Grant of Chongqing Medical University, China (Grant No CXTDG 201602007), and General Program of Health and Family Planning Commission of Chongqing, China (Grant No 2010-2-115). , 
 Retracted [22 June 2022] . ------------------ The Editor and Publisher of International Journal of Nanomedicine wish to retract the published article. Concerns were raised regarding the alleged duplication of images in Figure 6. Specifically, - Figure 6A, Non-targeted group, appears to have been duplicated with Figure 6A, Antagonized group. The authors responded to our queries and provided some of the original data relating to Figure 6. However, the Editor determined that current and previous concerns relating to this article have raised doubts about the validity of the findings and requested for the article to be retracted. The authors have been informed of this decision. Our decision-making was informed by our policy on publishing ethics and integrity and the COPE guidelines on retraction. The retracted article will remain online to maintain the scholarly record, but it will be digitally watermarked on each page as u201cRetractedu201d. , 
 #14 I thank the new editors for their efforts. ,