title: [Study on gene transfection in bone marrow mesenchymal stem cells mediated by plasmid of bone morphogenetic protein 2 loaded lipopolysaccharide-amine nanopolymersomes].
abstract: OBJECTIVE
  To evaluate the combination of lipopolysaccharide-amine nanopolymersomes (LNPs), as a gene vector, with target gene and the transfection in bone marrow mesenchymal stem cells (BMSCs) so as to provide a preliminary experiment basis for combination treatment of bone defect with gene therapy mediated by LNPs and stem cells.
  
  
  METHODS
  Plasmid of bone morphogenetic protein 2 (pBMP-2)-loaded LNPs (pLNPs) were prepared. The binding ability of pLNPs to pBMP-2 was evaluated by a gel retardation experiment with different ratios of nitrogen to phosphorus elements (N/P). The morphology ofpLNPs (N/P = 60) was observed under transmission electron microscope (TEM) and atomic force microscope (AFM). The size and Zeta potential were measured by dynamic light scattering (DLS). The resistance of pLNPs against DNase I degradation over time was explored. The viability of BMSCs, transfection efficiency, and expression of target protein were investigated after transfection by pLNPs in vitro.
  
  
  RESULTS
  At N/P ≥ 1.5, pLNPs could completely retard pBMP-2; at N/P of 60, pLNPs was uniform vesicular shape under AFM; TEM observation demonstrated that pLNPs were spherical nano-vesicles with the diameter of (72.07 ± 11.03) nm, DLS observation showed that the size of pLNPs was (123 ± 6) nm and Zeta potential was 20 mV; pLNPs could completely resist DNase I degradation within 4 hours, and such protection capacity to pBMP-2 decreased slightly at 6 hours. The cell survival rate first increased and then decreased with the increase of N/P, and reached the maximum value at N/P of 45; the cytotoxicity was in grade I at N/P ≤ 90, which meant no toxicity for in vivo experiment. While the transfection efficiency of pLNPs increased with the increase of N/P, and reached the maximum value at N/P of 60. So it is comprehensively determined that the best N/P was 60. At 4 days, transfected BMSCs expressed BMP-2 continuously at a relatively high level at N/P of 60.
  
  
  CONCLUSIONS
  LNPs can compress pBMP-2 effectively to form the nanovesicles complex, which protects the target gene against enzymolysis. LNPs has higher transfection efficiency and produces more amount of protein than polyethylenimine 25k and Lipofectamine 2000.
authors.affiliation: Department of Prosthodontics, guangdong Provincial Key Laboratory of Stomatology, Sun Yat-Sen University, Guangzhou Guangdon, P.R. China.
authors.forename: Yun
authors.initials: Y
authors.lastname: Guan
authors.forename: Qinmei
authors.initials: Q
authors.lastname: Wang
authors.forename: Ying
authors.initials: Y
authors.lastname: Cheng
authors.forename: Wei
authors.initials: W
authors.lastname: Teng
authors.forename: Hongzhang
authors.initials: H
authors.lastname: Huang
mesh_terms: D000588:Amines; D001854:Bone Marrow Cells / Q000166:cytology / Q000378:metabolism*; D055396:Bone Morphogenetic Protein 2 / Q000235:genetics / Q000378:metabolism*; D001842:Bone and Bones; D005786:Gene Expression Regulation; D015316:Genetic Therapy; D005822:Genetic Vectors; D006412:Hematopoietic Stem Cells; D006801:Humans; D008070:Lipopolysaccharides / Q000737:chemistry*; D059630:Mesenchymal Stem Cells / Q000187:drug effects / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011108:Polymers / Q000737:chemistry; D014162:Transfection
chemical_list: D000588:Amines; D055396:Bone Morphogenetic Protein 2; D008070:Lipopolysaccharides; D011108:Polymers