title: Preparation and evaluation of nanoparticles loading plasmid DNAs inserted with siRNA fragments targeting c-Myc gene.
abstract: BACKGROUND
  c-Myc plays a key role in glioma cancer stem cell maintenance. A drug delivery system, nanoparticles loading plasmid DNAs inserted with siRNA fragments targeting c-Myc gene (NPs-c-Myc-siRNA-pDNAs), for the treatment of glioma, has not previously been reported.
  
  
  OBJECTIVE
  NPs-c-Myc-siRNA-pDNAs were prepared and evaluated in vitro.
  
  
  METHODS
  Three kinds of c-Myc-siRNA fragments were separately synthesized and linked with empty siRNA expression vectors in the mole ratio of 3:1 by T4 DNA ligase. The linked products were then separately transfected into Escherichia coli. DH5α followed by extraction with Endofree plasmid Mega kit (Qiagen, Hilden, Germany) obtained c-Myc-siRNA-pDNAs. Finally, the recombinant c-Myc-siRNA3-pDNAs, generating the highest transfection efficiency and the greatest apoptotic ability, were chosen for encapsulation into NPs by the double-emulsion solvent-evaporation procedure, followed by stability, transfection efficiency, as well as qualitative and quantitative apoptosis evaluation.
  
  
  RESULTS
  NPs-c-Myc-siRNA3-pDNAs were obtained with spherical shape in uniform size below 150 nm, with the zeta potential about -18 mV, the encapsulation efficiency and loading capacity as 76.3 ± 5.4% and 1.91 ± 0.06%, respectively. The stability results showed that c-Myc-siRNA3-pDNAs remained structurally and functionally stable after encapsulated into NPs, and NPs could prevent the loaded c-Myc-siRNA3-pDNAs from DNase degradation. The transfection efficiency of NPs-c-Myc-siRNA3-pDNAs was proven to be positive. Furthermore, NPs-c-Myc-siRNA3-pDNAs produced significant apoptosis with the apoptotic rate at 24.77 ± 5.39% and early apoptosis cells observed.
  
  
  CONCLUSIONS
  Methoxy-poly-(ethylene-glycol)-poly-(lactide-co-glycolide) nanoparticles (MPEG-PLGA-NPs) are potential delivery carriers for c-Myc-siRNA3-pDNAs.
authors.affiliation: Department of Surgery, Shanghai Institute of Digestive Surgery, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine , Shanghai , China.
authors.forename: Tao
authors.initials: T
authors.lastname: Ma
authors.forename: Jin-Ling
authors.initials: JL
authors.lastname: Jiang
authors.forename: Ying
authors.initials: Y
authors.lastname: Liu
authors.forename: Zheng-Bao
authors.initials: ZB
authors.lastname: Ye
authors.forename: Jun
authors.initials: J
authors.lastname: Zhang
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000235:genetics; D045744:Cell Line, Tumor; D016503:Drug Delivery Systems; D004926:Escherichia coli / Q000235:genetics; D016259:Genes, myc / Q000235:genetics*; D005910:Glioma / Q000235:genetics* / Q000628:therapy; D053758:Nanoparticles; D010316:Particle Size; D010957:Plasmids; D011091:Polyesters / Q000737:chemistry; D011092:Polyethylene Glycols / Q000737:chemistry; D034741:RNA, Small Interfering / Q000008:administration & dosage*; D051381:Rats; D014162:Transfection
chemical_list: D011091:Polyesters; D034741:RNA, Small Interfering; C558447:methoxypolyethyleneglycol-poly(lactic-co-glycolic acid); D011092:Polyethylene Glycols
keywords: Apoptosis; MPEG–PLGA; double-emulsion solvent-evaporation; flow cytometry; glioma; transfection