title: Novel epithelial cell adhesion molecule antibody conjugated polyethyleneimine-capped gold nanoparticles for enhanced and targeted small interfering RNA delivery to retinoblastoma cells.
abstract: BACKGROUND
  Several nanoconjugates have been designed to deliver nucleic acids such as small interfering RNA (siRNA) and DNA to cells to study silencing and expression efficacies. In the present study, we prepared novel epithelial cell adhesion molecule (EpCAM) monoclonal antibody conjugated polyethyleneimine (PEI) capped gold nanoparticles (AuNPs) loaded with EpCAM-specific siRNA molecules to knock-down the EpCAM gene in retinoblastoma (RB) cells. We chose EpCAM as a target moiety to deliver siRNA because this molecule is highly expressed in various epithelial cancers and is an ideal target as it is highly expressed in the apical surface of tumor cells while showing basolateral expression in normal cells.
  
  
  METHODS
  The EpCAM antibody was conjugated to AuNP-PEI loaded with siRNA molecules to specifically deliver siRNA to EpCAM-expressing RB cells. Conjugation efficiencies were confirmed with ultraviolet-visible spectrophotometry, Fourier transform infrared spectroscopy, and agarose and SDS-polyacrylamide gel electrophoresis. The size and zeta potential were measured using a Zeta sizer analyzer. Nanoparticle internalization and uptake were studied using fluorescent microscopy and flow cytometry. Gene silencing efficacy was monitored with western blot analysis and real-time quantitative PCR.
  
  
  RESULTS
  Optimal size and neutral zeta potential properties of the AuNP-PEI- EpCAM antibody (EpAb) antibody were achieved for the transfection studies. The AuNP-PEI nanoparticles did not show any cytotoxicity to the cells, which means these nanomaterials are suitable for intracellular delivery of siRNA for therapeutic interventions. With EpCAM antibody conjugation, PEI-capped AuNPs loaded with EpCAM siRNA were significantly internalized in the Y79 cells as observed with fluorescence microscopy and flow cytometry and induced a highly significant reduction in the cell viability of the Y79 cells. Through increased binding of EpCAM antibody-conjugated AuNP-PEI nanoparticles, significant downregulation of EpCAM gene was observed in the Y79 cells when compared to the cells treated with the antibody-unconjugated AuNP-PEI nanoparticles.
  
  
  CONCLUSIONS
  Thus, a novel antibody conjugated nanocarrier designed to deliver siRNA holds promise as an effective gene therapy strategy for retinoblastoma in the near future. In addition to serving as an siRNA delivery tool for therapy, gold nanoparticles can also serve as imaging modality in diagnosis.
authors.affiliation: Department of Ocular Pathology, Vision Research Foundation, Sankara Nethralaya, Chennai, India.
authors.forename: Moutushy
authors.initials: M
authors.lastname: Mitra
authors.forename: Mallikarjuna
authors.initials: M
authors.lastname: Kandalam
authors.forename: Judith
authors.initials: J
authors.lastname: Rangasamy
authors.forename: Balaji
authors.initials: B
authors.lastname: Shankar
authors.forename: Uma K
authors.initials: UK
authors.lastname: Maheswari
authors.forename: Sethuraman
authors.initials: S
authors.lastname: Swaminathan
authors.forename: Subramanian
authors.initials: S
authors.lastname: Krishnakumar
mesh_terms: D000951:Antigens, Neoplasm / Q000276:immunology*; D015815:Cell Adhesion Molecules / Q000276:immunology*; D016923:Cell Death / Q000187:drug effects; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D004587:Electrophoresis, Agar Gel; D004705:Endocytosis / Q000187:drug effects; D000071858:Epithelial Cell Adhesion Molecule; D005434:Flow Cytometry; D020868:Gene Silencing / Q000187:drug effects; D018014:Gene Transfer Techniques; D006046:Gold / Q000737:chemistry*; D006801:Humans; D053768:Metal Nanoparticles / Q000737:chemistry* / Q000633:toxicity / Q000648:ultrastructure; D008856:Microscopy, Fluorescence; D010316:Particle Size; D011094:Polyethyleneimine / Q000737:chemistry*; D034741:RNA, Small Interfering / Q000008:administration & dosage* / Q000378:metabolism; D015203:Reproducibility of Results; D012175:Retinoblastoma / Q000235:genetics* / Q000473:pathology
chemical_list: D000951:Antigens, Neoplasm; D015815:Cell Adhesion Molecules; D000071858:Epithelial Cell Adhesion Molecule; D034741:RNA, Small Interfering; D006046:Gold; D011094:Polyethyleneimine