title: Efficient downregulation of VEGF in retinal pigment epithelial cells by integrin ligand-labeled liposome-mediated siRNA delivery.
abstract: BACKGROUND
  The purpose of this study was to demonstrate the effectiveness of an integrin peptide ligand-labeled liposomal delivery system loaded with vascular endothelial growth factor (VEGF)-siRNA in a model study of gene therapy for retinopathy using human retinal pigment epithelial cells.
  
  
  METHODS
  Arg(R)-Gly(G)-Asp(D) motif peptide conjugating polyethylene glycol modified (RGD-PEGylated) liposomes were prepared using a thin-film hydration method and optimized for surface charge, particle size, small interfering RNA (siRNA) load, and entrapment efficiency. Reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assays were used to determine VEGF levels in retinal pigment epithelial cells. Cytotoxicity was determined using the 3-[4, 5-dimethylthiazol-2-yl]-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay and flow cytometry.
  
  
  RESULTS
  Physicochemical properties, including particle size, zeta potential, and siRNA load, of the prepared RGD-PEGylated liposomes and their entrapment efficiency were determined to be within the following ranges: 123.8-234.1 nm, 17.31-40.09 m V, 5.27%-6.33%, and >97%, respectively. RGD-PEGylated liposome-mediated fluorescent-labeled siRNA delivery demonstrated significantly enhanced cellular uptake, and 3 mol% RGD-PEGylated liposomes (having 3β-[N-(N', N'-dimethylaminoethane) carbamoyl] cholesterol (DC-cholesterol) DSPE and DSPE-PEG(2000)-RGD with molar ratio of 50/47/3) were shown to have better efficacy with regard to specificity for retinal pigment epithelial cells, reduced cytotoxicity, and knockdown of the target molecule.
  
  
  CONCLUSIONS
  By integrin receptor-mediated endocytosis, 3 mol% RGD-PEGylated liposomes were shown to be a suitable vector when loaded with VEGF-siRNA for efficient downregulation of VEGF in retinal pigment epithelial cells at both the protein and gene levels. This integrin ligand-modified liposomal delivery system has therapeutic potential for ocular gene therapy.
authors.affiliation: Chengwei Biotechnology Co, Ltd., Taipei, Taiwan.
authors.forename: Cheng-Wei
authors.initials: CW
authors.lastname: Chen
authors.forename: Ming-Kung
authors.initials: MK
authors.lastname: Yeh
authors.forename: Chia-Yang
authors.initials: CY
authors.lastname: Shiau
authors.forename: Chiao-Hsi
authors.initials: CH
authors.lastname: Chiang
authors.forename: Da-Wen
authors.initials: DW
authors.lastname: Lu
mesh_terms: D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D015536:Down-Regulation / Q000187:drug effects*; D005434:Flow Cytometry; D006801:Humans; D016023:Integrins / Q000378:metabolism; D042541:Intracellular Space; D008081:Liposomes / Q000737:chemistry* / Q000494:pharmacology / Q000633:toxicity; D018613:Microscopy, Confocal; D010316:Particle Size; D020033:Protein Isoforms / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D055213:Retinal Pigment Epithelium / Q000166:cytology*; D042461:Vascular Endothelial Growth Factor A / Q000032:analysis / Q000235:genetics / Q000378:metabolism*
chemical_list: D016023:Integrins; D008081:Liposomes; D020033:Protein Isoforms; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D042461:Vascular Endothelial Growth Factor A
keywords: liposome; retinal pigment epithelial cells; siRNA delivery; vascular endothelial growth factor
references.citation: Invest Ophthalmol Vis Sci. 1998 Aug;39(9):1703-12
references.pmid: 9699560
references.citation: J Control Release. 2011 Dec 10;156(2):203-11
references.pmid: 21839126
references.citation: Ophthalmologica. 2007;221(6):411-7
references.pmid: 17947829
references.citation: J Pharm Sci. 2006 Sep;95(9):1856-72
references.pmid: 16850395
references.citation: Adv Drug Deliv Rev. 2009 Aug 10;61(10):850-62
references.pmid: 19422869
references.citation: Gene Ther. 2009 May;16(5):645-59
references.pmid: 19194480
references.citation: Adv Drug Deliv Rev. 1997 Aug 11;27(1):17-28
references.pmid: 10837548
references.citation: Biochemistry. 1998 Sep 15;37(37):12875-83
references.pmid: 9737866
references.citation: J Control Release. 2011 Jul 30;153(2):141-8
references.pmid: 21447361
references.citation: Angiogenesis. 2007;10(2):103-17
references.pmid: 17340211
references.citation: Int J Nanomedicine. 2011;6:1017-25
references.pmid: 21720513
references.citation: Proc Natl Acad Sci U S A. 1996 Sep 3;93(18):9764-9
references.pmid: 8790405
references.citation: Nature. 2009 Jan 22;457(7228):426-33
references.pmid: 19158789
references.citation: Biochim Biophys Acta. 2002 Aug 31;1564(2):343-8
references.pmid: 12175916
references.citation: Invest Ophthalmol Vis Sci. 2009 Jul;50(7):3438-46
references.pmid: 19255151
references.citation: J Control Release. 2009 Dec 3;140(2):157-65
references.pmid: 19686788
references.citation: Int J Pharm. 2009 Dec 1;382(1-2):254-61
references.pmid: 19666094
references.citation: Retrovirology. 2005 Oct 18;2:63
references.pmid: 16232320
references.citation: Diabetes. 2006 Feb;55(2):312-7
references.pmid: 16443762
references.citation: PLoS One. 2008;3(11):e3554
references.pmid: 18978936
references.citation: Prog Retin Eye Res. 2008 Jul;27(4):331-71
references.pmid: 18653375
references.citation: J Control Release. 2006 Jun 28;113(2):155-63
references.pmid: 16764960
references.citation: Nat Rev Drug Discov. 2009 Feb;8(2):129-38
references.pmid: 19180106
references.citation: Bioconjug Chem. 2000 May-Jun;11(3):425-32
references.pmid: 10821660
references.citation: Am J Pathol. 1999 Aug;155(2):421-8
references.pmid: 10433935
references.citation: Expert Opin Drug Deliv. 2005 Mar;2(2):237-54
references.pmid: 16296751
references.citation: Am J Pathol. 2005 Nov;167(5):1451-9
references.pmid: 16251428
references.citation: J Comp Neurol. 1995 Sep 11;360(1):1-16
references.pmid: 7499556
references.citation: AAPS PharmSciTech. 2008;9(3):798-809
references.pmid: 18597175
references.citation: Pathobiology. 2010;77(2):88-95
references.pmid: 20332668
references.citation: Cell. 2002 Sep 20;110(6):673-87
references.pmid: 12297042
references.citation: Nat Rev Drug Discov. 2007 Jun;6(6):443-53
references.pmid: 17541417
references.citation: Int J Nanomedicine. 2012;7:4861-72
references.pmid: 23028215
references.citation: Biomaterials. 2008 Aug-Sep;29(24-25):3477-96
references.pmid: 18499247
references.citation: Mol Ther. 2010 Apr;18(4):828-34
references.pmid: 20068551
references.citation: Invest Ophthalmol Vis Sci. 2008 Oct;49(10):4523-7
references.pmid: 18441313
references.citation: J Control Release. 2002 Jan 17;78(1-3):259-66
references.pmid: 11772466
references.citation: Int J Pharm. 2010 May 10;390(2):198-207
references.pmid: 20116418
references.citation: Development. 2012 Apr;139(8):1371-80
references.pmid: 22434866
references.citation: Vaccine. 2008 Apr 24;26(18):2225-33
references.pmid: 18396362
references.citation: Cancer Res. 1992 Jun 15;52(12):3255-61
references.pmid: 1596882
references.citation: Int J Nanomedicine. 2011;6:2567-80
references.pmid: 22128247