Mechanisms Of The Singlet Oxygen-Triggered Disassembly Of Hypoxia-Responsive Polymeric Micelles

Hypoxia-sensitive nanomicelles are on-demand nanocarriers that can be used for the triggered delivery of anti-cancer drugs to enhance anti-tumor therapy.The drug release profile depends on the extent of hypoxia within the solid tumor tissues.However,the oxygen level in solid tumors is not homogenous,which makes it difficult to fully release the power of such stimuli-responsive delivery vehicles.In order to solve this problem,a ¹O₂-responsive and hypoxia-sensitive micellear nonacarrier was designed.Upon photo-triggering,the swelling of micelles could enhance the liberation of encapsulated photosensitizers.Nitroimidazole?NI?,as a well-known hypoxic-sensitive small molecule,could be reduced to amino imidazole with the aid of nitroreductases that are over-expressed in a hypoxic environment,thereby enabling the transition from hydrophobicity to hydrophilicity.NI was covalently linked to the backbone of hydrophobic segment via an amide method.The model photosensitize Chlorin e6?Ce6?was physically encapsulated in the hydrophobic core of the amphiphilic copolymer micelle.The photosensitizer-loaded micelles were sensitive to light irradiation that could trigger the generation of ¹O₂.The produced ¹O₂ further induces the conversion of the hydrophobic nitroimidazole to hydrophilic oxalic aldehyde,which in turn caused a dramatic size expansion ofmicellar nanocarrier,leading to the rapid release and intracellular distribution of Ce6.The successful synthesis of the responsive copolymer?mPEG-PAsp-NI?and non-responsive control copolymer?mPEG-PBLA?was verified by proton nuclear magnetic resonance spectroscopy and fourier transform infrared spectroscopy.The critical micelle concentration?CMC?was determined using a fluorescent pyrene probe and the calculated value was 12.0±0.3?g/mL.Upon nitroimidazole reacting with singlet oxygen,the intermediates and final products were analyzed by mass spectrometry,which indirectly proved the proposed mechanism.One of the reaction products was oxalic aldehyde that could undergo an aldehyde group specific reaction and produce significant fluorescence absorption.For both free NI?and NI polymer,the maximum extent of oxidation?was ca.65%under the experimental settings of the current study.The hydrodynamic size and morphology of polymer micelles were assessed via dynamic light scattering and transmission Electron Microscope?TEM?,respectively.The results showed that the size of RM/Ce6 micelles increased with increasing irradiation time up to 50 minutes.In contrast,when the photosensitizer was absent,the micelles'hydrodynamic diameter remained constant before and after light irradiation.The TEM results were consistent with the DLS analysis.The drug loading of the micelles was determined by high performance liquid chromatography at 3.7%?w/w?in RM micelles and 1.0%?w/w?in the CM micelles.The release experiments were performed at pH 7.4 with or without light treatment The cumulative cargo release from RM was only 21%after 36 h under dark,whereas the corresponding value was 54%with laser treatment.This was because that the nitroimidazole in mPEG-PAsp-NI could react with the ¹O₂ produced by the photosensitizer upon laser irradiation,resulting in a change in the polymer's amphiphilicity,micelle disassembly,and hence accelerated Ce6 release.In the absence of light,there was no ¹O₂ in the system and the conversion of imidazole to oxalic aldehyde is not possible.Therefore,the micelles'hydrodynamic size remained constant,and there was no significant difference in drug release between CM and RM.This size-expandable nanosystem could deliver more Ce6 into 4T1 tumor cells accompanied with enhanced cytotoxicity.In summary,we have successfully discovered the singlet oxygen sensitivity of nitroimidazole and developed dually hypoxia-and singlet oxygen-sensitive polymer micelles.This novel nanoplatform shows potential application in the combinational delivery of photosensitizer with the traditional cytotoxic active agents.