title: A novel covalent approach to bio-conjugate silver coated single walled carbon nanotubes with antimicrobial peptide.
abstract: BACKGROUND
  Due to increasing antibiotic resistance, the use of silver coated single walled carbon nanotubes (SWCNTs-Ag) and antimicrobial peptides (APs) is becoming popular due to their antimicrobial properties against a wide range of pathogens. However, stability against various conditions and toxicity in human cells are some of the major drawbacks of APs and SWCNTs-Ag, respectively. Therefore, we hypothesized that APs-functionalized SWCNTs-Ag could act synergistically. Various covalent functionalization protocols described previously involve harsh treatment of carbon nanotubes for carboxylation (first step in covalent functionalization) and the non-covalently functionalized SWCNTs are not satisfactory.
  
  
  METHODS
  The present study is the first report wherein SWCNTs-Ag were first carboxylated using Tri sodium citrate (TSC) at 37 °C and then subsequently functionalized covalently with an effective antimicrobial peptide from Therapeutic Inc., TP359 (FSWCNTs-Ag). SWCNTs-Ag were also non covalently functionalized with TP359 by simple mixing (SWCNTs-Ag-M) and both, the FSWCNTs-Ag (covalent) and SWCNTs-Ag-M (non-covalent), were characterized by Fourier transform infrared spectroscopy (FT-IR), Ultraviolet visualization (UV-VIS) and transmission electron microscopy (TEM). Further the antibacterial activity of both and TP359 were investigated against two gram positive (Staphylococcus aureus and Streptococcus pyogenes) and two gram negative (Salmonella enterica serovar Typhimurium and Escherichia coli) pathogens and the cellular toxicity of TP359 and FSWCNTs-Ag was compared with plain SWCNTs-Ag using murine macrophages and lung carcinoma cells.
  
  
  RESULTS
  FT-IR analysis revealed that treatment with TSC successfully resulted in carboxylation of SWCNTs-Ag and the peptide was indeed attached to the SWCNTs-Ag evidenced by TEM images. More importantly, the present study results further showed that the minimum inhibitory concentration (MIC) of FSWCNTs-Ag were much lower (~7.8-3.9 µg/ml with IC50: ~4-5 µg/ml) compared to SWCNTs-Ag-M and plain SWCNTs-Ag (both 62.6 µg/ml, IC50: ~31-35 µg/ml), suggesting that the covalent conjugation of TP359 with SWCNTs-Ag was very effective on their counterparts. Additionally, FSWCNTs-Ag are non-toxic to the eukaryotic cells at their MIC concentrations (5-2.5 µg/ml) compared to SWCNTs-Ag (62.5 µg/ml).
  
  
  CONCLUSIONS
  In conclusion, we demonstrated that covalent functionalization of SWCNTs-Ag and TP359 exhibited an additive antibacterial activity. This study described a novel approach to prepare SWCNT-Ag bio-conjugates without loss of antimicrobial activity and reduced toxicity, and this strategy will aid in the development of novel and biologically important nanomaterials.
authors.affiliation: Center for Nanobiotechnology Research, Alabama State University, Montgomery, AL, USA.
authors.forename: Atul A
authors.initials: AA
authors.lastname: Chaudhari
authors.affiliation: Center for Nanobiotechnology Research, Alabama State University, Montgomery, AL, USA.
authors.forename: D'andrea
authors.initials: D
authors.lastname: Ashmore
authors.affiliation: Department of Mechanical Engineering, University of Louisville, Louisville, KY, USA.
authors.forename: Subrata Deb
authors.initials: SD
authors.lastname: Nath
authors.affiliation: Department of Mechanical Engineering, University of Louisville, Louisville, KY, USA.
authors.forename: Kunal
authors.initials: K
authors.lastname: Kate
authors.affiliation: Center for Nanobiotechnology Research, Alabama State University, Montgomery, AL, USA.
authors.forename: Vida
authors.initials: V
authors.lastname: Dennis
authors.affiliation: Center for Nanobiotechnology Research, Alabama State University, Montgomery, AL, USA.
authors.forename: Shree R
authors.initials: SR
authors.lastname: Singh
authors.affiliation: Therapeutic Peptides Inc., 7053 Revenue Drive, Baton Rouge, LA, 70809, USA.
authors.forename: Don R
authors.initials: DR
authors.lastname: Owen
authors.affiliation: Therapeutic Peptides Inc., 7053 Revenue Drive, Baton Rouge, LA, 70809, USA.
authors.forename: Chris
authors.initials: C
authors.lastname: Palazzo
authors.affiliation: Department of Drug Discovery and Development, Auburn University, Auburn, AL, USA.
authors.forename: Robert D
authors.initials: RD
authors.lastname: Arnold
authors.affiliation: Research Instrumentation Facility, Auburn University, Auburn, AL, USA.
authors.forename: Michael E
authors.initials: ME
authors.lastname: Miller
authors.affiliation: Center for Nanobiotechnology Research, Alabama State University, Montgomery, AL, USA. spillai@alasu.edu.
authors.forename: Shreekumar R
authors.initials: SR
authors.lastname: Pillai
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000737:chemistry* / Q000494:pharmacology; D023181:Antimicrobial Cationic Peptides / Q000737:chemistry* / Q000494:pharmacology; D002460:Cell Line; D045744:Cell Line, Tumor; D002951:Citrates / Q000737:chemistry; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects; D004926:Escherichia coli / Q000187:drug effects / Q000254:growth & development; D006801:Humans; D008264:Macrophages / Q000166:cytology / Q000187:drug effects; D051379:Mice; D008826:Microbial Sensitivity Tests; D037742:Nanotubes, Carbon / Q000737:chemistry* / Q000648:ultrastructure; D012486:Salmonella typhimurium / Q000187:drug effects / Q000254:growth & development; D012834:Silver / Q000737:chemistry* / Q000494:pharmacology; D013211:Staphylococcus aureus / Q000187:drug effects / Q000254:growth & development; D013297:Streptococcus pyogenes / Q000187:drug effects / Q000254:growth & development
chemical_list: D000900:Anti-Bacterial Agents; D023181:Antimicrobial Cationic Peptides; D002951:Citrates; D037742:Nanotubes, Carbon; C514290:trisodium citrate; D012834:Silver
keywords: Antimicrobial; Bacteria; Bio-conjugation; Carbon nanotubes; Cytotoxicity; Peptide