Efficient Generation And Fluorescence Detection Of Singlet Oxygen Based On Polymeric Hybrid Nanocarriers

Photodynamic therapy(PDT)is an emerging technology based on the interaction of light,oxygen and photosensitizer for the treatment of tumors or other diseases.Photosensitizers were activated with light of a specific wavelength,consequently the excited photosensitizers transfer their energy to O2,then generating a large number of reactive oxygen species(ROS)mainly composed of singlet oxygen(1O2).And 1O2 can oxidize the organelles and bio-macromolecules in cells,resulting in cell damage and the apoptosis or necrosis of tumor cells.Compared with traditional treatment,such as surgical,chemotherapy and radiotherapy,PDT with the advantages of minimal invasiveness,highly selectivity and rarely side effects,has attracted lots of attention from many researchers.However,the quantum yield of 1O2 is also limited by the aggregation of photosensitizers and tumor hypoxia.What's more,monitoring of the production and distribution of intracellular 1O2 during PDT in real time is also beneficial for assessing the efficacy of PDT and achieving precise personalized treatment.Therefore,the nanophotosensitizer and fluorescent nanoprobe based on porous and gas permeable polymer-organic silica hybrid nanoparticles were respectively constructed for efficient generation and fluorescence detection of 1O2 during PDT in real time.The main contents and results are shown as follows:1.Synthesis and optimization of ZnPc-loaded polymeric hybrid NPs(nanoparticles)for efficient PDT.Different concentration of the second-generation photosensitizer of ZnPc were incorporated into NPs by using reprecipitation-encapsulation method(PS and DTS as the matrix),then the NPs were further modified with a thick poly-L-lysine(PLL)layer owing to electrostatically interaction(ZnPc-PS@SiO2@PLL-NPs).The positively charged PLL shell not only endows the NPs with good biocompatibility and efficient cellular uptake but also effectively prevents the leakage of ZnPc.In addition,the nanophotosensitizers(ZnPc-PVK@SiO2@PLL-NPs and ZnPc-PFO@SiO2@PLL-NPs)were prepared by using PVK(Poly(N-vinylcarbazole))and PFO(Poly(9,9-dioctylfluorenyl-2,7-diyl))as polymer matrix.The relationship between the degree of aggregation(DOA)of ZnPc in NPs and the doping concentration,and the effect of DOA on the yield of 1O2 were investigated.The results showed that:(1)For the three types of NPs,the optimal doping concentration of ZnPc is 4%,at which point the NPs have an optimal 1O2 generation rate;(2)the DOA of ZnPc in three types of NPs is different:ZnPc molecules hold the largest degree of aggregation in PS@SiO2@PLL NPs and the lowest quantum yield of 1O2,while the ZnPc molecules own the lowest degree of aggregation in PFO@SiO2@PLL-NPs and the highest quantum yield of 1O2.The results indicated that a polymer with a large rigid structural unit(rigid structure size:PS<PVK<PFO)can effectively alleviate the aggregation of ZnPc in the NPs,thereby increasing the yield of 1O2.And 4 wt%ZnPc-PFO@SiO2@PLL-NPs showed excellent inhibition on the growth of tumor cells for both in vitro and in vivo PDT.2.Preparation of fluorinated nanophotosensitizers with self-supplied oxygen for efficient PDT.In the light of the excellent oxygen affinity of perfluorocarbons(PFCs)and the presence of fluorine can improve the photo-and oxidative stability of the photosensitizers,the fluorinated nanophotsensitizers(ZnPcF16-PFDTS-NPSs)were constructed for efficient PDT by using PS as the polymer matrix and using perfluorodecyltriethoxysilane(PFDTS)and fluorine-substituted ZnPc(ZnPcF16)as the substitutes for DTS and ZnPc.The optimal doping concentration of ZnPcF16 is 4wt%.And compared with non-fluorinated nanophotosensitizers(ZnPcF 16-DTS-NPSs),ZnPcF16-PFDTS-NPSs have higher dissolved oxygen content,improved 1O2 quantum yield and better in vitro PDT effect,which is mainly due to the fact that PFDTS provides a perfluorocarbon chain with oxygen carrying capacity for nanoparticles.3.Two types of fluorescent nanoprobes were prepared for real-time monitoring of intracellular 1O2 during PDT.Type ?:The commercial hydrophobic 1O2-probe 1,3-diphenylisobenzofuran(DPBF)molecules were encapsulated into the polymeric hybrid NPs(DPBF-PS-NPs)to detect 1O2 by using the fluorescence characteristics of DPBF(?ex=405 nm,?em=455 nm)towards 1O2.20wt%DPBF doped NPs were used to monitor the generation of intracellular 1O2 during PDT,then the fluorescence of DPBF is quenched by 1O2.Owing to the hydrophobicity of polymeric core,the other ROS(·OH,O2·-,H2O2,etc.)cannot get in or out of the nanoprobes,but O2 and 1O2(in the form of gas)can.That not only protects the DPBF from being quenched by other ROS,but also ensure their specificity.Type ?:In order to improve the photostability of nanoprobes,we used two-photon excited congjugated polymer PFO(?ex=800 nm,?em=441 nm)as the matrix and designed a kind of two-photon excited fluorescence enhanced nanoprobes(DPBF-PFO-NPs)to detect 1O2 based on the fluorescence resonance energy transfer between PFO and DPBF.When the DPBF doping concentration is 20 wt%,the fluorescence of PFO(50wt%)at 441 nm is almost quenched under 800 nm excitation(two-photon).With the generation of 1O2,DPBF molecules were consumed,and the fluorescence of PFO at 441 nm is gradually increased.The photostability of the nanoprobes has been greatly improved.And the nanoprobes also hold the same specificity and show highly detection sensitivity with the limit of detection(LOD,?350 nM).Both types of probes can monitor the generation of intracellular 1O2 during PDT in real time by fluorescence imaging,and provide support for accurate and effective photodynamic therapy.