1. cyclic peptide-polymer conjugates synthesis.
A cyclic peptide was designed with azido and amino groups on opposite sides, to provide a platform for an orthogonal azide-alkyne ‘click’ reaction and amidation reaction. N-hydroxysuccinimide (NHS) functionalized polymer pNBMA with controlled molecular weight was synthesized by reversible addition−fragmentation chain-transfer (RAFT) polymerization and conjugated in slight excess to the cyclic peptide by mild amidation reaction. The product was easily purified by precipitation in dichloromethane/diethyl ether mixture to remove the excess pNBMA. Then a pPEGA with bicyclo-[6.1.0]-nonyne (BCN) as chain-end was obtained by modifying the NHS-pPEGA with a commercially available strained alkyne, and further conjugated, in slight excess, to the cyclic peptide by a catalyst free strain promoted azide-alkyne cycloaddition. pNBMA25-CP-pPEGA27 was obtained by precipitation in methyl tert-butyl ether to remove unreacted BCN-pPEGA.
2. Self-assembly of cyclic peptide-polymer conjugates
Transmission electron microscopy (TEM) and scanning electron microscope (SEM) were used to verify the morphology of the cyclic peptide-induced self-assembly of the conjugates. Unlike the corresponding block copolymer pNBMA25-b-pPEGA27, which self-assembles into typical spherical micelles with a diameter of 27 ± 2 nm driven by hydrophobic-hydrophilic interactions in water. pNBMA25-CP-pPEGA27 self-assembled into distinctly different cylindrical structures with an average length of 129 ± 23 nm and width of 25 ± 3 nm. Small angle neutron scattering (SANS) showed the system was assembling as hairy cylindrical micelle models, which is in good agreement with the observed morphology from TEM and SEM.
3. Photo-responsive property of obtained tubisomes
UV-vis spectroscopy was first employed to monitor the photo-cleavage process of the self-assembled tubisomes . Upon irradiation with 365 nm UV light, the characteristic absorption band for o-nitrobenzyl ester group at 320 nm decreased, accompanied by the increase of absorbance at 360 nm (characteristic absorption for o-nitrosobenzaldehyde), demonstrating the photo-stimulated cleavage of the pNBMA segment. SANS and TEM were employed. As indicated by TEM, all the tubular structures disappeared after irradiation with 365 nm UV light for 20 minutes, indicating the disassembly of the tubisomes.Fitting the data collected from SANS after irradiation revealed that the nanotube structures had disappeared and only Gaussian chains remain in solution.
4. Drug loading and toxicity
The toxicity of pNBMA25-CP-pPEGA27 was investigated by 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT) assay on human breast cancer MDA-MB-231 cells. Even if the concentration of pNBMA25-CP-pPEGA27 reached up to 500 μg/mL, the cell survival rates are still higher than 90%, indicating low cytotoxicity of the tubisomes. Upon deprotonating doxorubicin hydrochloride with trimethylamine, DOX was successfully loaded into the hydrophobic core of tubisomes by co-self-assembly in water. On the basis of the standard absorbance curve for DOX, the drug loading content of tubisomes was calculated at 10.3 wt.%. Without photo stimulus, only 13% of encapsulated DOX was released within 24 h, indicating a good load-retention capability of the tubisome carriers under physiological conditions. However, a much faster release of DOX was observed after photo-irradiating the DOX-loaded tubisomes solution for 20 minutes. The release increased to 60% within 24 h, demonstrating the photo-triggered dissociation of the supramolecular tubisomes indeed enhanced the drug release.
5. Cellular uptake and release
Owning to the intrinsic fluorescence of DOX, the cellular uptake and intracellular release of DOX-loaded tubisomes can be traced by confocal laser scanning microscopy (CLSM).
