Pharmaceutical Micelles Featured With Singlet Oxygen-Responsive Cargo Release And Mitochondrial Targeting For Enhanced Photodynamic Therapy

The combination of smart controlled nanocarrier system with photodynamic therapy?PDT?has been widely employed in cancer treatment.However,the efficiency of nanoparticle photodynamic therapy is often compromised by the short life and limited diffusion radius of singlet oxygen as well as uncontrolled intracellular distribution of photosensitizer.It was hypothesized that the rapid photosensitizer release upon nanoparticle internalization and its preferred accumulation in mitochondria would address the above problems.Hence,the aim of this study was to engineeramultifunctionalmicellarnanosystemfeaturedwithsinglet oxygen-responsive cargo release and mitochondria targeting.An imidazole-bearing amphiphilic copolymer was employed as the micelles building block to encapsulate triphenylphosphonium-pyropheophorbide a?TPP-PPa?conjugate or PPa.The TPP-PPa conjugate was encapsulated in the ¹O₂-responsive micelles by a modified oil-in-water emulsion-based self-assembly approach.The multifunctional micelles were used to control the drug release and target mitochondrial for improved the anti-cancer PDT efficiency.Upon laser irradiation,the singlet oxygen produced by TPP-PPa/PPa oxidized the imidazole moiety to produce hydrophilic urea,leading to micelle disassembly and rapid cargo release.The particle size and morphology of each drug-loaded micelle were investigated by dynamic light scattering?DLS?and transmission electron microscope?TEM?.The results showed that the size of micelle increased with increasing of irradiation time.The drug loading was determined by high performance liquid chromatography?HPLC?.The drug content in the two kinds of micelles?mPEG-PAsp-IM@PPa and mPEG-PAsp-IM@TPP-PPa?were 1.8±0.1%and 3.1±0.2%?w/w?.Furthermore,the singlet oxygen quantum yield of the TPP-PPa was measured at 0.38 which showed that the TPP-PPa could be a potential pontent of photosensitizer.Regarding the in vitro release experiment,the experimental group and the control group were treated with laser and non-laser respectively under pH 7.4 and the cumulative release of laser-free micelles in the experimental group was 27.3±1.6%after 36 hours.However,the cumulative release reached 49.1±2.7%with laser after 36 h.Then the results of MTT assay in 4T1 cells showed that the mPEG-PAsp-IM@TPP-PPa micelles have higher toxicity and the IC₅₀0 was 2.1±0.2?M.Meanwhile,the cellular uptake results revealed that TPP-PPa primarily accumulated in the mitochondria and lead to mitochondrial disruption and cell apoptosis.The co-localization analysis showed that the TPP moiety significantly enhanced the photosensitizer uptake in mitochondria,improved mitochondria depolarization upon irradiation,and hence boosted the cytotoxicity in 4T1 cells.Irrespective of the irradiation conditions,both Pearson's correlation coefficient?PCO?and Mander's overlap coefficient?MOC?ranked as follows:free TPP-PPa>TPP-PPa micelles>free PPa>PPa micelles.The targeting strategy also dramatically reduced the intracellular ATP level as a consequence of mitochondria injury.The mitochondria damage was accompanied with the activation of the apoptosis signals?Caspase 3 and Caspase 9?,whose level was directly correlated to the apoptosis extent.Meanwhile,the live/dead cell staining indicated that the viability ranked as follows:free TPP-PPa>TPP-PPa micelles>free PPa>PPa micelles,which agreed with the cytotoxicity data.To sum up,this thesis reported a new idea for improving the efficiency of anti-tumour PDT by loading mitochondria-targeted TPP-PPa in ¹O₂-responsive micelles.This work provides a facile and robust means to enhance the efficacy of photodynamic therapy.