Effect of integrin receptor-targeted liposomal paclitaxel for hepatocellular carcinoma targeting and therapy
Authors: Kai Liu
Liyu Chen
Yanbin Liu
Weiya Wang
Journal: Oncology Letters (2015)
Discipline: CLINICAL MEDICINE
Keywords: integrin receptor; liposome; paclitaxel; tumor targeting.
Affiliations: Center of Infectious Diseases, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, P.R. China., Department of Pathology, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, P.R. China.

Abstract:  The major aim of the present study was to develop an integrin receptor-targeted liposomal paclitaxel (PTX) to enhance the targeting specificity and therapeutic effect of PTX on hepatocellular carcinoma (HCC) cells. The specific Arg-Gly-Asp (RGD) ligand was conjugated to 1,2-distearoylphosphatidylethanolamine-polyethylene glycol 2000 to prepare the RGD-modified liposomes (RGD-LP). Furthermore, physicochemical characteristics of RGD-LP, including particle size, ζ potential, encapsulation efficiency and in vitro PTX release, were evaluated. RGD-modified liposomes were selected as the carrier for the present study, as they exhibit good biocompatibility and are easy to modify using RGD. The cellular uptake efficacy of RGD-LP by HepG2 cells was 3.3-fold higher than that of liposomes without RGD, indicating that RGD-LP may specifically target HepG2 cells by overexpressing integrin αvβ3 receptors. The RGD modification appeared to enhance the anti-proliferative activity of LP-PTX against HepG2 cells, with the extent of anti-proliferative activity dependent on the concentration of PTX and the incubation time. Additionally, evaluation of the homing specificity and anticancer efficacy of RGD-LP on the tumor spheroids indicated that solid tumor penetration was enhanced by the modification of RGD. In agreement with these in vitro findings, in vivo investigations demonstrated that RGD-LP-PTX exhibited a greater inhibitory effect on tumor growth in HepG2-bearing mice than LP-PTX or free PTX. Thus, RGD-LPs may represent an efficient targeted PTX delivery system for the treatment of patients with HCC. 

Retracted: Yes

PubPeer comments:
 Numerous textual similarities between this paper and Wang et al. (2014),  [Efficacy of dual-functional liposomes containing paclitaxel for treatment of lung cancer]  . For instance, the present second paragraph: blockquote Over the last two decades, liposomal drug delivery systems have exhibited significant potential for the delivery of therapeutic agents to tumors, and various strategies have been used to improve their targeting specificity and cellular uptake. For example, polyethylene glycol (PEG)ylation has been extensively employed to increase the accumulation of liposomes in tumor tissues via enhanced permeability and retention (EPR) effects (i.e., passive targeting) (11). To increase the specificity of the interactions between liposomes and tumor cells, various studies have focused on the development of active tumor-targeted liposomes modified with specific ligands, including transferrin (12), folic acid (13), peptides (14u201318) or antibodies (19u201321). These modified liposomes are able to selectively recognize and bind to specific receptors overexpressed on tumor cells. Clinically, these modifications are able to increase targeting efficiency and reduce toxicity. blockquote and the corresponding second paragraph of Wang et al.: blockquote In the last two decades, liposomal drug delivery systems hold extraordinary potential for delivery of therapeutics to tumor, and various strategies have been used to improve their targeting specificity and cellular uptake. PEGylation has been extensively employed to enhance the accumulation of liposomes in tumor tissues through enhanced permeability and retention (EPR) effects, which was the passive form of targeting (3). In attempts to increase the specificity of interaction between liposomes and tumor cells, recent efforts in the liposome field have focused on the development of active tumor-targeted liposomes, which were modified with specific ligands such as TF (4), folic acid (5), peptides (6u20139) or antibodies (10u201312), and could selectively recognize and bind to the specific overexpressed receptor in tumor cells, resulting in increased targeting efficiency and less toxicity. blockquote , 
 Materials and methods: blockquote Soybean lecithin consisting of 90u201395% phosphatidylcholine (SPC), and 1,2-distearoylphosphatidylethanolamine methylated polyethylene glycol 2000 (DSPE-mPEG2000), and DSPE-PEG2000-Maleimide (Mal) were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). Additionally, cholesterol was purchased from Chengdu Kelong Chemical Co. Ltd. (Chengdu, China) and carboxyfluorescein phosphatidylethanolamine (CFPE) was purchased from Avanti Polar Lipids, Inc. The RGD peptide with a terminal cysteine was synthesized according to the standard solid phase peptide synthesis protocol described by the manufacturer (Shanghai Jier Bio-Pharmaceutical Co., Ltd., Shanghai, China). Furthermore, DAPI was obtained from Beyotime Institute of Biotechnology (Haimen, China), 1,10-dioctadecyl-3,3, 30,30-tetramethylindotricarbocyanineiodide was purchased from Biotium, Inc. (Hayward, CA, USA) and cell culture plates were purchased from Wuxi Nest Biotechnology Co., Ltd. (Wuxi, China). All other chemicals and reagents were of analytical grade and obtained commercially. BALB c male athymic nude mice (weight, ~20 g; n=40) were purchased from the Experimental Animal Center of Sichuan University (Chengdu, China). blockquote Wang et al.: blockquote Soybean lecithin consisting of 90u201395% phosphatidylcholine and mPEG2000-DSPE, and Mal-PEG2000-DSPE were purchased from Avanti Lipid (Alabaster, AL, USA). Cholesterol (CHO) was purchased from Chengdu Kelong Chemical Company (Chengdu, China). Rhodamine-PE was purchased from Avanti Lipid. T7 peptide with terminal cysteine (Cys-HAIYPRH) and TAT peptide with terminal cysteine (Cys-AYGRKKRRQRRR) were produced according to the standard solid phase peptide synthesis by Shanghai Jier Bio-Pharmaceutical Co., Ltd. (Shanghai, China). Cell culture plates were purchased from Wuxi NEST Biotechnology Co., Ltd. (Wuxi, China). Other chemicals and reagents were of analytical grade and obtained commercially. BALB c male athymic nude mice (~20 g) were purchased from the Experiment Animal Center of Shandong University (Jinan, China). blockquote , 
 Figure 1: ![file] appears to have been assembled from the same graphic components, presumably by the same illustrator, as Figure 1 of Wang et al.: ![file] , 
 Conclusion: blockquote In conclusion, the present study successfully developed tumor-targeting liposomes modified with the integrin receptor-specific RGD ligand (RGD-LP-PTX). This liposomal delivery system exhibited increased cellular uptake efficiency and targeting specificity in HepG2 cells with high integrin receptor expression levels, compared with PTX alone or LP-PTX, which are two commonly used paclitaxel dosage forms within the clinic. In addition, efficient targeted delivery of the therapeutic agent was achieved in HepG2 tumor cells and HepG2 tumor-bearing nude mice, ultimately achieving high therapeutic efficacy in the tumor-bearing mice. Based on the investigations conducted in the current study, it was hypothesized that RGD-modified liposomes may be used as a potential anti-tumor PTX delivery system for the effective treatment of patients with HCC. blockquote Wang et al.: blockquote In conclusion, in this study, we successfully developed the dual-ligand liposomes modified with the specific ligand T7 motif and non-specific TAT. This liposomal delivery system possessed increased cellular uptake efficiency and targeting specificity in A549 cells whose TFR expression levels were high, and achieved an efficient synergistic targeted delivery of payload into tumor cells in A549 tumor bearing nude mice, ultimately achieving excellent therapeutic efficacy on tumor-bearing mice. The findings of this study suggest that the T7- and TAT-modified liposomes are a potential antitumor drug delivery system. blockquote , 
 Wu et al. (2014),  [Development of synthetic of peptide-functionalized liposome for enhanced targeted ovarian carcinoma therapy]  (e-Century publishing) appear to be following the same script, albeit applying it to a different cancer. For comparison with comment #1: blockquote In the last two decades, liposomal drug delivery systems hold extraordinary potential for delivery of therapeutics to tumor, various strategies have been used to improve their targeting specificity and cellular uptake. PEGylation has been extensively employed to enhance the accumulation of liposomes in tumor tissues through enhanced permeability and retention (EPR) effects, which was the passive form of targeting. In attempts to increase the specificity of interaction between liposomes and tumor cells, recent efforts in the liposome field have been focusing on the development of the active tumor targeted liposomes, which were modified with some specific ligands such as Tf [12], folic acid [13], peptides [14] or antibodies [15], and could selectively recognize and bind to the specific receptor over-expressed on tumor cells, resulting in increased targeting efficiency and less toxicity. blockquote For comparison with comment #2: blockquote Soybean lecithin consisting of 90-95% phosphatidylcholine and mPEG2000-DSPE and Mal-PEG2000-DSPE were purchased from Avanti lipid (USA). Cholesterol (CHO) was purchased from Chengdu Kelong Chemical Company (Chengdu, China). FITC-PE was purchased from Avanti lipid (USA). T7 peptide with terminal cysteine (Cys-HAIYPRH) was synthesized according to the standard solid phase peptide synthesis by Shanghai Dacheng Bio-pharmaceutical Co., Ltd. (Shanghai, China). Cell culture plates were purchased from corning Biotechnology Co. Ltd. (USA). Other chemicals and reagents were of analytical grade and obtained commercially. BALB c female athymic mice (about 20 g) were purchased from the Experiment Animal Center of Zhengzhou University (P. R. China). blockquote For comparison with comment #4: blockquote In conclusion, we successfully developed the targeting liposomes modified with the specific ligand T7. This liposomal delivery system possessed increased cellular uptake efficiency and targeting specificity in A2780 cells whose transferrin expression levels were high, and achieved an efficient targeted delivery of payload into tumor cells in A2780 tumor bearing nude mice and ultimately achieved excellent therapeutic efficacy on tumor-bearing mice. Based on all the studies in this report, we claimed the T7 modified liposomes as a potential anti-tumor drug delivery system. blockquote No overlap between author lists or institutions, so I am forced to speculate that all three papers were the work of the anonymous staff of a paper-mill. , 
 The present Figure 9: ![file] is clearly the same results as Figure 9 from Wang et al. ![file] Evidently liposomal paclitaxel inhibits the growth of HepG2 and A549 xenographs in nude mice in b exactly the same way b . , 
 #6 I was just saying to my colleagues.  The problem is we will not be able to spot this in graphs . Unless you do it this way of course. Good that you found this. , 
 It is not the same. In the upper Figure 9  relative tumor volume is measured, so treatment with PBS is the worst, whereas in the lower Figure 9,  tumor inhibition rate is measured, and PBS inhibits tumor growth the most, so it is the best treatment. That said, the similarity of these unrelated experiments measuring different tumors in different ways do look surprisingly similar. , 
 #8 Let s say that identical **results** are being plotted (as can be shown by overlying one chart on the other), except that Wang et al. plot two additional conditions. Although we are informed that **entirely different research teams** collected these identical results, measuring **different variables**, with **different xenograft metastases**. , 
 #7 *  The problem is we will not be able to spot this in graphs .* There is no substitute for the pattern-matching skills of an experienced art historian. , 
 ![file] Here are the relationships I ve found. The references are: Cao Y, Zhou Y, Zhuang Q, Cui L, Xu X, Xu R, He X. Anti-tumor effect of RGD modified PTX loaded liposome on prostatic cancer. Int J Clin Exp Med. 2015 Aug 15;8(8):12182-91. eCollection 2015. PubMed PMID: 26550128; PubMed Central PMCID: PMC4612813. Chen L, Liu Y, Wang W, Liu K. Effect of integrin receptor-targeted liposomal paclitaxel for hepatocellular carcinoma targeting and therapy. Oncol Lett. 2015 Jul;10(1):77-84. Epub 2015 May 20. PubMed PMID: 26170980; PubMed Central PMCID: PMC4487032. Guo Y, Wang L, Lv P, Zhang P. Transferrin-conjugated doxorubicin-loaded lipid-coated nanoparticles for the targeting and therapy of lung cancer. Oncol Lett. 2015 Mar;9(3):1065-1072. Epub 2014 Dec 30. PubMed PMID: 25663858; PubMed Central PMCID: PMC4315058. Liu Y, Ran R, Chen J, Kuang Q, Tang J, Mei L, Zhang Q, Gao H, Zhang Z, He Q. Paclitaxel loaded liposomes decorated with a multifunctional tandem peptide for glioma targeting. Biomaterials. 2014 Jun;35(17):4835-47. doi: 10.1016 j.biomaterials.2014.02.031. Epub 2014 Mar 17. PubMed PMID: 24651033. Mei L, Fu L, Shi K, Zhang Q, Liu Y, Tang J, Gao H, Zhang Z, He Q. Increased tumor targeted delivery using a multistage liposome system functionalized with RGD, TAT and cleavable PEG. Int J Pharm. 2014 Jul 1;468(1-2):26-38. doi: 10.1016 j.ijpharm.2014.04.008. Epub 2014 Apr 5. PubMed PMID: 24709209 Tang J, Zhang L, Gao H, Liu Y, Zhang Q, Ran R, Zhang Z, He Q. Co-delivery of doxorubicin and P-gp inhibitor by a reduction-sensitive liposome to overcome multidrug resistance, enhance anti-tumor efficiency and reduce toxicity. Drug Deliv. 2016 May;23(4):1130-43. doi: 10.3109 10717544.2014.990651. Epub 2014 Dec 10. PubMed PMID: 25491241. Wang RH, Cao HM, Tian ZJ, Jin B, Wang Q, Ma H, Wu J. Efficacy of dual-functional liposomes containing paclitaxel for treatment of lung cancer. Oncol Rep. 2015 Feb;33(2):783-91. doi: 10.3892 or.2014.3644. Epub 2014 Dec 2. PubMed PMID: 25482610. Wu H, Yao L, Mei J, Li F. Development of synthetic of peptide-functionalized liposome for enhanced targeted ovarian carcinoma therapy. Int J Clin Exp Pathol. 2015 Jan 1;8(1):207-16. eCollection 2015. PubMed PMID: 25755707; PubMed Central PMCID: PMC4348858. Zheng C, Ma C, Bai E, Yang K, Xu R. Transferrin and cell-penetrating peptide dual-functioned liposome for targeted drug delivery to glioma. Int J Clin Exp Med. 2015 Feb 15;8(2):1658-68. eCollection 2015. PubMed PMID: 25932094; PubMed Central PMCID: PMC4402741. , 
 #6 Here s that graph again! Though this time the units are cubic-mm rather than  relative volume % : ![file] Also the tumors this time are **PC-3** tumor xenografts. Source: Cao et al (2015),  [Anti-tumor effect of RGD modified PTX loaded liposome on prostatic cancer]  (Int. J. Clin. Exp. Med). , 
 March 2021 retraction: https: www.spandidos-publications.com 10.3892 ol.2021.12611 From the notice:  *Subsequently to the publication of the above paper, it has been brought to the attention of the Editor that there were a number of striking similarities between the article and another paper published in a different journal by different authors located at different research institutions. First, numerous textual similarities were noted between several paragraphs of the text comparing between this paper and the other paper, which was published in Oncology Reports, also in 2015: Wang R-H, Cao H-M, Tian Z-J, Jin B, Wang Q, Ma H and Wu J: Efficacy of dual-functional liposomes containing paclitaxel for treatment of lung cancer. Oncol Rep 33: 783u2013791, 2015. Furthermore, Fig. 9 in the above article contained data plots that were also strikingly similar to those included in Fig. 9 in the paper published in Oncology Reports.* *After having considered these aspects, the Editor of Oncology Letters has decided that the above paper should be retracted from the publication on the grounds of a lack of certainty regarding the independence and integrity of the study. The authors were contacted in this regard, but did not offer any rebuttal in light of the Editor s decision to retract this paper. Note that the study by Wang et al in Oncology Reports has also been retracted. The Editor regrets any inconvenience that this retraction has caused.* ,