Development of Novel Drug and Gene Delivery Carriers Composed of Single-Walled Carbon Nanotubes and Designed Peptides With PEGylation

Abstract

Single-walled carbon nanotubes (SWCNTs) attract great interest in biomedical applications including drug and gene delivery. In this study, we developed a novel delivery system using SWCNTs associated with designed polycationic and amphiphilic peptides. Wrapping of SWCNTs with H-(-Lys-Trp-Lys-Gly-)7-OH [(KWKG)7] resulted in stable dispersion in water, but the composite aggregated in the buffered solution. This dispersion instability was also evident in a cell culture medium with fetal bovine serum. To improve the aqueous dispersibility, the SWCNTs-(KWKG)7 composite was further modified with polyethylene glycol (PEG) at the lysine residues via amide bond formation and the highest modification extent of 13.3% of the amino groups which corresponded to 2 PEG chains in each peptide molecule was achieved with fluorescein isothiocyanate-labeled carboxyl-PEG12. The uptake of the SWCNTs composite by A549 human lung adenocarcinoma epithelial cells was evaluated by visual observation and fluorescence activated cell sorting analysis for SWCNTs wrapped with a mixture of (KWKG)7 with PEGylation and H-(-Cys-Trp-Lys-Gly-)-OH-(KWKG)6 [CWKG(KWKG)6] labeled with fluorescent boron-dipyrromethene tetramethylrhodamine and 7-fold higher uptake comparing with SWCNTs-peptide composite without PEGylation was obtained suggesting the importance of dispersibility in addition to a cationic charge. The superior potential of SWCNTs composites assisted by polycationic and amphiphilic peptides with PEGylation was thus demonstrated.

Sustained Release of Mitomycin C from Its Conjugate with Single-Walled Carbon Nanotubes Associated by Pegylated Peptide

“…5). The pegylated SWCNTs-CWKG(KWKG) 6 -(PEG) 12 exhibited superior dispersion stability in an aqueous solution and cell culture medium for 24 h by visual observation, like SWCNTs-(KWKG)7-(PEG) 12 complex reported previously 17) (data not shown). Synthesis of MMC-EMCA The aziridine group of MMC was transformed with EMCA by EDC reaction for affording a linker with the maleimide group necessary for conjugation to the thiol group of SWCNTs-CWKG(KWKG) 6 -(PEG) 12 .…”

“…Thus, the difference of an amino acid between CWKG(KWKG) 6 and (KWKG) 7 did not significantly affect the PEG modification efficiency and/or the physical or chemical properties. Since the cellular uptake of SWCNTs-(KWKG) 7 -(PEG) 12 was verified, 17) cellular delivery of SWCNTs-CWKG(KWKG) 6 -(PEG) 12 was also expected, suggesting its potential as a DDS carrier. The present development of a sustained release device for MMC utilizing the SWCNTs-CWKG(KWKG) 6 -(PEG) 12 is a prototype research for the expansion of the possibility of SWCNTs-peptide-PEG composites as well as the extension of previous studies that focused on macromolecular prodrugs of MMC.…”

“…The reaction was performed for 2 h, and the reaction solution was filtered by centrifugation three times using an Amicon Ultra-0.5 Centrifugal Filter 100 kD Device (Merck Millipore Ltd., Germany) for exchanging the buffer and removing free unconjugated MS(PEG) 12 . The extent of PEG modification of amino groups was determined as reported in a previous study 17) using fluorescein isothiocyanate (FITC)-(PEG) 12 -NHS ester and was calculated from the absorbance spectrum and molar extinction coefficient of FITC (494 nm and A 494 nm =68000 M −1 cm…”

“…The SWCNTs-CWKG(KWKG) 6 composite was prepared in 5 mL D 2 O solution containing 2 mg SWCNTs and 10 mg CWKG(KWKG) 6 peptide in a glass tube by sonicating for 1 h with an ultrasonic disruptor UD-201 (TOMY Digital Biology, Tokyo, Japan) in an ice water bath. 11,17) The chemical and structural stability of peptides during sonication was demonstrated in our previous study, where a complex formation of peptides with SWCNTs was indicated by atomic force microscopy (AFM) image of SWCNTs-peptide and β-sheet formation of peptide was assigned by circular dichroism (CD) spectrum.…”

“…Moreover, polyethylene glycol (PEG) modifications of the SWCNTs-(KWKG) 7 composites remarkably improved the dispersion stability even under a biological environment, thereby suggesting the tremendous potential of these composites as drug and gene carriers. 17) In this study, SWCNTs composites with CWKG(KWKG) 6 peptides having thiol and amino groups were employed and MMC was conjugated to the thiol groups employing N-ε-maleimidocaproic acid (EMCA) as a linker to transform the aziridine group of MMC after pegylation of amino groups. The controlled release characteristics and cytotoxic activities of the SWCNTs-CWKG(KWKG) 6 -PEG 12 -C 6 -MMC composites are discussed.…”