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Tubular Supramolecular Polymer Brushes as Novel Therapeutic Materials

Periodic Reporting for period 1 - TSPBNTM (Tubular Supramolecular Polymer Brushes as Novel Therapeutic Materials)

Reporting period: 2017-09-01 to 2019-08-31

The morphology of self-assembled nanostructures play a very important role in their functionalities, for instance, cylindrical micelles have been reported to show increased in vivo circulation times and different cellular internalisation pathways compared to spherical nanoparticles, thus making them a promising platform for biomedical applications. Here we report that introducing highly directional and robust supramolecular interactions enables to transform the self-assembly of block copolymers from spherical into rigid cylindrical micelles (tubisomes). In our system, cyclic peptides (CPs), flat ring-like peptides consisting of an even number of alternating D- and L-amino acids, were synthesized and employed as a joint linker between the hydrophilic and hydrophobic segments of amphiphilic block copolymers. The multiple hydrogen bonding interactions between the CPs can drive the formation of tubular nanostructures. As a result, the amphiphilic CP-polymer conjugates self-assemble into tubisomes independent of the ratio of the hydrophilic and hydrophobic domains, thereby providing an efficient approach for cylindrical polymeric micelles. Moreover, accessible modifications of either cyclic peptides or conjugated polymers could endow the tubisomes with a host of functionalities, which can make them versatile candidates for various applications especially as drug delivery vehicles.
To this end, we further exploit the potential of tubisomes as controlled drug delivery systems. Photo-responsive tubisome was fabricated to realise the controlled release of anticancer drug Doxorubicin, and the anticancer efficacy was evaluated. Furthermore, to get better understanding of tubisomes based drug delivery vehicles, fluorescent labelling which can track their intracellular distribution during drug delivery is our concern. By synergetic combination of aggregation induced emission effects and cyclic peptide shaped tubular self-assembly, we reported the construction of a type of multi-functional tubisome in water, which shows unique topological structure and excellent fluorescent property. In-situ monitoring of drug release was realised. This work provides an efficient method to prepare cylindrical polymeric micelles and an intriguing drug delivery mode which can determine the intracellular location of the delivery vehicles and the drug releasing position, showing promising potentials in cancer therapy.
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).
1. The related projects will be finished within the next two months and will be summarised into manuscripts to submit to high impact factor journals.( one manuscript submittd to Angewandte chemi is under review)
2.Our finding paves an alternative way for fabricating polymeric tubular structures and expands the toolbox for the rational design of functional hierarchical nanostructures, collaborations will be established with school of life science and medical school to further explore the applications in cancer therapy.