Synthesis Of Novel Phthalocyanine Based Nanophotosensitizers And Their Application In Organelles Labels And Fluorescence Imaging-guided In Vitro Photodynamic Therapy

Photodynamic therapy(PDT)is a minimally invasive and safty method for cancers.It has attracted increasing attention owing to its precise controllable treatment and low skin toxicity.Phthalocyanines exhibit high singlet oxygen producing ability,stable and easily modification structure,thereby they are a promising second generation photosensitizer.However,their poor solubility and limited ability to target tumor had limited their application in PDT.The design and synthesis imaging-guided phthalocyanine photosensitizers with organelle targeting has become a research hotspot.In this paper,a series of phthalocyanine photosensitizers and their water-soluble nanoparticles were designed and synthesized,and their photophysical and photochemical properties,two-photon fluorescence imaging properties,organelle localization in breast cells as well as in vitro photodynamic activity were studied.(1)A cholesterol silicon(?)phthalocyanine(Chol-Pc)and its water soluble Chol-Pc based nanoparticle(DSPE@Chol-Pc)were synthesized.DSPE@Chol-Pc is spherical with a mean diameter of 66 nm.The Q-band absorption of Chol-Pc and DSPE@Chol-Pc are located at 673 nm and 689 nm,resepectively.Chol-Pc exhibited aggregation in nanoparticles,the Q-band of DSPE@Chol-Pc was red-shift,and the fluorescence intensity and the fluorescence quantum yield decreased,and the fluorescence lifetime was shortened.The trafficking of DSPE@Chol-Pc in breast cancer cells was visualized by tracking the fluorescence of Chol-Pc and FITC labeled DSPE-mPEG₂₀₀₀ through two-photon imaging in real-time.It was founded that Chol-Pc disassociated from the DSPE-mPEG₂₀₀₀ on the plasma membrane and travelled to the cholesterol-rich domains.Besides,both Chol-Pc and DSPE@Chol-Pc could effectively produce reactive oxgen species in MCF-7 and MDA-MB-231 cells and exhibited excellent phototoxicity effects on breast cancer cell,leading to the cell death through destroy the function of cholesterol-rich membrane system.(2)Silicon/zinc phthalocyanines with triphenylamine substituted naming:bis-(4'-diphenylamino-[1,1'-biphenyl]-4-ylmethoxy)phthalocyninesilicon(?)(TPA-SiPc)and tetra-?-(4'-diphenylamino-[1,1'-biphenyl]-4-ylmethoxy)phthalocynine zinc(?)(TPA-ZnPc)were synthesized and characterized.TPA-SiPc and TPA-ZnPc exhibited similar absorption spectra and equivalent fluorescence quantum yields,fluorescence lifetimes and singlet oxygen quantum yields.The fluorescence spectra of TPA-SiPc and TPA-ZnPc in different proportion of THF/H₂O solution are different:the emission peak for TPA at 400 nm and for SiPc at 672 nm were observed for TPA-SiPc when the water ratio of THF/H₂O solution was up to 60%,However,the emission peak of TPA for TPA-ZnPc increased while the fluorescence of ZnPc at 687nm decreased sharply with the increase of H₂O ratio,which was related to the different solubility and stacking patterns of triphenylamine substituted silicon/zinc phthalocyanines at different proportions of H₂O/THF.The water-soluble nanoparticles DSPE@TPA-SiPc and DSPE@TPA-ZnPc were prepared using DSPE-mPEG₂₀₀₀ as a nanocarrier.DSPE@TPA-SiPc and DSPE@TPA-ZnPc are spherical with particle sizes of 60 nm and 87 nm respectively.Compared with free TPA-SiPc and TPA-ZnPc,the absorption and fluorescence emission of DSPE@TPA-SiPc and DSPE@TPA-ZnPc were red-shifted,the absorption and fluorescence intensity were decreased,and the fluorescence lifetimes were shortened.Under the irradiation of 670nm plus 730 nm,the SiPc and TPA moieties of DSPE@TPA-SiPc exhibited the cooperative singlet oxygen production ability.It was found that DSPE@TPA-SiPc could simultaneously target lipid droplets and lysosomes by co-localization with the lipid droplets and lysosomes commercial dyes.Upon the irradiation with 670 nm plus730 nm laser,DSPE@TPA-SiPc-mediated photodynamic therapy can synergally kill MCF-7 and MDA-MB-231 breast cells with the killing rate of more than 85%.(3)Trifluoromethyl substituted pyrrolidone silicon/zinc phthalocyanine naming:(Z/E)-(bis-4-(N-(2-(3,4-dicyanophenoxy)ethyl)oxopyrrolidin-3-ylidene-2,2,2-trifluoro ethyl))phthalocyaninesilicon(?)(PyCF₃SiPc),(Z/E)-Tetra-?-4-(2-(3-(1-chloro-2,2,2-trifluoroethylidene)oxopyrrolidinyl)ethoxy)phth alocyanineZinc(PyCF₃ZnPc)and(Z/E)-tetra-?-4-(2-(3-(2,2,2-trifluoro-(4-diphenylaminophenyl)ethylidene)oxopyrrolid inyl)ethoxy)phthalocynine zinc(?)(PyCF₃TPAZnPc)were synthesized and their structures were characterized.The Q-band of PyCF₃SiPc,PyCF₃ZnPc and PyCF₃TPAZnPc are located at 674-679 nm,and PyCF₃SiPc,PyCF₃ZnPc and PyCF₃TPAZnPc mainly exsited as monomer.PyCF₃SiPc had the strongest singlet oxygen generation capacity and fluorescence lifetime.The water-soluble nanoparticles DSPE@PyCF₃SiPc and TPA-mPEG@PyCF₃SiPc were prepared using DSPE-mPEG₂₀₀₀ and TPA-mPEG₂₀₀₀ as nanocarriers,respectively.The particle sizes of DSPE@PyCF₃SiPc and TPA-mPEG@PyCF₃SiPc were 86 nm and 60 nm respectively.DSPE@PyCF₃SiPc and TPA-mPEG@PyCF₃SiPc could produce singlet-oxygen species in aqueous solution.DSPE@PyCF₃SiPc and TPA-mPEG@PyCF₃SiPc could specifically target at lipid droplets.The dynamic movement of lipid droplets could be tracked by DSPE@PyCF₃SiPc and TPA-mPEG@PyCF₃SiPc.DSPE@PyCF₃SiPc and TPA-mPEG@PyCF₃SiPc exhibited low dark toxicity and strong phototoxicity to the MCF-7 breast cancer cells.Under light irradiation,they can induce cell death by destroying lipid droplets function of the breast cancer cells.(4)Morpholinate and pyrrolidonate substituted silicon phthalocyanines:di(4'-(2-(oxopyrrolidinyl)ethoxycarbonyl)-[1,1'-biphenyl]-4-yl)phthalocynine silicon(?)(PySiPc)and di(4'-(2-(morpholinoyl)ethoxycarbonyl)-[1,1'-biphenyl]-4-yl)phthalocynine silicon(?)(MSiPc)were synthesized and characterized.They were mainly exist as monomers.The absorption spectra,fluorescence quantum yields,singlet-oxygen quantum yields as well as fluorescence lifetimes are similar.DSPE-mPEG₂₀₀₀ was used as a nanocarrier to encapsulate PySiPc and MSiPc to form DSPE@PySiPc and DSPE@MSiPc nanoparticles by a co-solvent method,DSPE@PySiPc and DSPE@MSiPc are spherical with a mean diameter of 70 nm and 88 nm.DSPE@PySiPc and DSPE@MSiPc could produce reactive oxygen species both in water and in breast cancer cell.DSPE@PySiPc and DSPE@MSiPc specifically target to the lysosomes of the MCF-7 and MDA-MB-231 breast cancer cell.Under light irradiation,DSPE@PySiPc and DSPE@MSiPc can produce reactive oxgen species,and damaged the lysosome leading to the death of cells.(5)The AIE fluorescence probes with pyrrolidone and morpholine substituted triphenylamine/tetrastyrene were synthesized and characterized.The Stokes shifts of TPE-Ma and TPE-Pywere about 200 nm and that of TPA-Ma,TPA-Py,TPA-2Pyand TPA-2Ma were also about 150 nm.The large Stokes shifts were beneficial for fluorescence imaging because of high signal-to-noise ratios.The fluorescent probes exhibited AIE effect in different proportion of H₂O/DMSO solution.DSPE-mPEG₂₀₀₀was used as a nanocarrier to encapsulate above AIE fluorescence probes to form nanoparticles.The particle sizes of nanoparticles were within 100 nm.Nanoparticles exhibited almost no dark toxicity to MCF-7 cells.TPE-Ma and TPE-Pyused as lysosomal probes,while TPA-Ma,TPA-Pyand TPA-2Ma used as lysosomes and lipid drop probes.