title: Comparing the intracellular fate of components within a noncovalent streptavidin nanoparticle with covalent conjugation.
abstract: BACKGROUND
  Auger radiotherapy requires adequate tumor delivery and high nuclear accumulation and retention. We hypothesize that the noncovalent nature of a streptavidin/biotin three-component nanoparticle possessing these qualities may be required for dissociation of the radiolabeled oligomer and its accumulation into the cell nucleus.
  
  
  METHODS
  As a test of our hypothesis, the intracellular fate of an antisense oligomer when incubated as the nanoparticle and when incubated while covalently conjugated to the antibody was compared. The three-component noncovalent nanoparticle consisted of streptavidin linking three biotinylated components: a Cy3-labeled anti-RIα antisense phosphorodiamidate morpholino (MORF) oligomer, a tat transfecting peptide and the anti-Her2 herceptin antibody. The covalent constructs included an anti-RIα antisense DNA conjugated to a radiolabeled herceptin and a fluorescent DNA conjugated to native herceptin. Fluorescence microscopy in SK-BR-3 (Her2+) cells was used to evaluate the fate of the fluorescent Cy5.5-DNA and Cy3-MORF, while the subcellular accumulation of the (111)In-labeled herceptin and herceptin-DNA in both SK-BR-3 and MDA-MB-231 (Her2) cells was determined by isolating and counting the nuclear fractions.
  
  
  RESULTS
  Previously, we demonstrated that when incubated as the three-component nanoparticle consisting of herceptin and streptavidin and (99m)Tc-labeled antisense MORF, only the MORF accumulated in the nucleus of Her2+ cells. In this investigation, clear evidence was observed of nuclear accumulation of the antisense oligomer within the noncovalent nanoparticle as before, but when incubated as the covalent construct, by both fluorescence microscopy and nuclear counting, no evidence of nuclear accumulation was observed.
  
  
  CONCLUSIONS
  The weaker noncovalent biotin-streptavidin bond may be essential for adequate delivery of the radiolabeled antisense oligomer to the nucleus of tumor cells.
authors.affiliation: Department of Radiology, University of Massachusetts Medical School, Worcester, MA 01655, USA.
authors.forename: Yuxia
authors.initials: Y
authors.lastname: Liu
authors.forename: Dengfeng
authors.initials: D
authors.lastname: Cheng
authors.forename: Xinrong
authors.initials: X
authors.lastname: Liu
authors.forename: Guozheng
authors.initials: G
authors.lastname: Liu
authors.forename: Shuping
authors.initials: S
authors.lastname: Dou
authors.forename: Nan
authors.initials: N
authors.lastname: Xiao
authors.forename: Ling
authors.initials: L
authors.lastname: Chen
authors.forename: Mary
authors.initials: M
authors.lastname: Rusckowski
authors.forename: Donald J
authors.initials: DJ
authors.lastname: Hnatowich
mesh_terms: D061067:Antibodies, Monoclonal, Humanized / Q000008:administration & dosage / Q000493:pharmacokinetics*; D000970:Antineoplastic Agents / Q000008:administration & dosage / Q000493:pharmacokinetics*; D001710:Biotin / Q000008:administration & dosage / Q000493:pharmacokinetics*; D045744:Cell Line, Tumor; D016503:Drug Delivery Systems / Q000379:methods; D005434:Flow Cytometry; D006801:Humans; D007202:Indicators and Reagents / Q000494:pharmacology; D007205:Indium Radioisotopes / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D053758:Nanoparticles; D009369:Neoplasms / Q000378:metabolism*; D019809:Streptavidin / Q000008:administration & dosage / Q000493:pharmacokinetics*; D000068878:Trastuzumab; D014803:Vitamin B Complex / Q000008:administration & dosage / Q000493:pharmacokinetics*
chemical_list: D061067:Antibodies, Monoclonal, Humanized; D000970:Antineoplastic Agents; D007202:Indicators and Reagents; D007205:Indium Radioisotopes; D014803:Vitamin B Complex; D001710:Biotin; D019809:Streptavidin; D000068878:Trastuzumab