A novel covalent approach to bio-conjugate silver coated single walled carbon nanotubes with antimicrobial peptide.
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).


CONCLUSION
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.
['Chaudhari|Atul A|AA|', "Ashmore|D'andrea|D|", 'Nath|Subrata Deb|SD|', 'Kate|Kunal|K|', 'Dennis|Vida|V|', 'Singh|Shree R|SR|', 'Owen|Don R|DR|', 'Palazzo|Chris|C|', 'Arnold|Robert D|RD|', 'Miller|Michael E|ME|', 'Pillai|Shreekumar R|SR|']
D000818:Animals D000900:Anti-Bacterial Agents D023181:Antimicrobial Cationic Peptides D002460:Cell Line D045744:Cell Line, Tumor D002951:Citrates D004847:Epithelial Cells D004926:Escherichia coli D006801:Humans D008264:Macrophages D051379:Mice D008826:Microbial Sensitivity Tests D037742:Nanotubes, Carbon D012486:Salmonella typhimurium D012834:Silver D013211:Staphylococcus aureus D013297:Streptococcus pyogenes
Bio-conjugation Carbon nanotubes Antimicrobial Peptide Bacteria Cytotoxicity