title: A nonviral pHEMA+chitosan nanosphere-mediated high-efficiency gene delivery system.
abstract: The transport of DNA into eukaryotic cells is minimal because of the cell membrane barrier, and this limits the application of DNA vaccines, gene silencing, and gene therapy. Several available transfection reagents and techniques have been used to circumvent this problem. Alternatively, nonviral nanoscale vectors have been shown to bypass the eukaryotic cell membrane. In the present work, we developed a unique nanomaterial, pHEMA+chitosan nanospheres (PCNSs), which consisted of poly(2-hydroxyethyl methacrylate) nanospheres surrounded by a chitosan cationic shell, and we used this for encapsulation of a respiratory syncytial virus (RSV)-F gene construct (a model for a DNA vaccine). The new nanomaterial was capable of transfecting various eukaryotic cell lines without the use of a commercial transfection reagent. Using transmission electron microscopy, (TEM), fluorescence activated cell sorting (FACS), and immunofluorescence, we clearly demonstrated that the positively charged PCNSs were able to bind to the negatively charged cell membrane and were taken up by endocytosis, in Cos-7 cells. Using quantitative polymerase chain reaction (qPCR), we also evaluated the efficiency of transfection achieved with PCNSs and without the use of a liposomal-based transfection mediator, in Cos-7, HEp-2, and Vero cells. To assess the transfection efficiency of the PCNSs in vivo, these novel nanomaterials containing RSV-F gene were injected intramuscularly into BALB/c mice, resulting in high copy number of the transgene. In this study, we report, for the first time, the application of the PCNSs as a nanovehicle for gene delivery in vitro and in vivo.
authors.affiliation: Center for NanoBiotechnology Research, Alabama State University, Montgomery, AL 36104, USA.
authors.forename: Erdal
authors.initials: E
authors.lastname: Eroglu
authors.forename: Pooja M
authors.initials: PM
authors.lastname: Tiwari
authors.forename: Alain B
authors.initials: AB
authors.lastname: Waffo
authors.forename: Michael E
authors.initials: ME
authors.lastname: Miller
authors.forename: Komal
authors.initials: K
authors.lastname: Vig
authors.forename: Vida A
authors.initials: VA
authors.lastname: Dennis
authors.forename: Shree R
authors.initials: SR
authors.lastname: Singh
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D019556:COS Cells; D048271:Chitosan / Q000008:administration & dosage / Q000737:chemistry*; D002522:Chlorocebus aethiops; D004337:Drug Carriers / Q000008:administration & dosage / Q000737:chemistry*; D005260:Female; D018628:Gene Dosage; D051379:Mice; D008807:Mice, Inbred BALB C; D054118:Nanospheres / Q000008:administration & dosage / Q000737:chemistry*; D011102:Polyhydroxyethyl Methacrylate / Q000008:administration & dosage / Q000737:chemistry*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger; D022261:Respiratory Syncytial Virus Vaccines / Q000235:genetics; D012136:Respiratory Syncytial Viruses / Q000235:genetics; D014162:Transfection / Q000379:methods*; D019444:Vaccines, DNA / Q000235:genetics; D014709:Vero Cells; D014759:Viral Envelope Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D004337:Drug Carriers; D012333:RNA, Messenger; D022261:Respiratory Syncytial Virus Vaccines; D019444:Vaccines, DNA; D014759:Viral Envelope Proteins; D011102:Polyhydroxyethyl Methacrylate; D048271:Chitosan
keywords: RSV-DNA vaccine; nonviral vector; pHEMA+chitosan nanoparticles