Development Of An Advanced Fluorescent Probe With Aggregation-Induced Emission Characteristics For Two-photon Photodynamic Therapy

Background&ObjectiveAs a non-invasive treatment with spatiotemporal controllability and low toxic side effects,photodynamic therapy(PDT)has been gradually applied to the treatment of tumors,condyloma and other diseases.Especially,two-photon PDT(TP-PDT)has been one of the most prominent strategies in imaging-guided therapy(IGT)due to its deep tissue penetration,high spatial selectivity as well as negligible damages to surrounding normal tissues.However,idea two-photon photosensitizers are still highly desirable.It’s have been reported that to develop small-molecule two-photon PSs with aggregation-induced emission(AIE)characteristics is a promising choice for TP-PDT.In this contribution,we firstly designed and synthesized an advanced small-molecule AIE probe with unique photophysical properties,namely DPASP.The aim of the second part is to study the performance of DPASP in cell imaging and its capacity to work as a PS.And in the third and forth part,the aim is to investigate the performance of DPASP in TP-PDT.Methods1.DPASP was synthesized following the route depicted in scheme 1.The structure of DPASP was fully characterized and verified by NMR and HRMS measurement and its photophysical properties were studied by UV-absorption spectrum analysis,photoluminescence(PL)analysis,and the detection of two-photon absorption cross section.2.The intracellular organelle-targeting capability of DPASP was studied by colocalization experiment and its photostability was detected using A549 cell line.Caspase-3 activation detection,CCK8 assay and reactive oxygen species(ROS)detection were performed to determine the capacity of DPASP to work as a PS.3.To verify the capacity of DPASP to work as a two-photon PS,A549 cells were co-incubated with DPASP and propidium iodide(PI),which is a common DNA-targeting probe used to indicate apoptosis,and scanned using two-photon laser.A549 cells were cultured into multicellular tumor spheroids(MCTSs)and co-stained with DPASP and PI.For stereotactic damage,the two-photon focal plane was fixed at the appointed depth in the MCTS and continuously sequential scanning was performed.The stereotactic function of DPASP-induced TP-PDT was assessed according to the distribution of PI-positive cells.4.A549 tumor-bearing nude mice model was established.The tumor retention property of DPASP was assessed by animal imaging experiments and the tissue penetration was detected by two-photon imaging.Finally,A549 tumor-bearing nude mice were randomly divided into different groups and each one was treated by intratumoral injection with DPAS(or PBS)and TP-PDT(or not).The tumor volumes were monitored and recorded after treatments.Results1.DPASP brings a large Stokes shift of 189 nm with both AIE and twisted intramolecular charge transfer(TICT)properties and show strong two-photon absorption capability with a large two-photon absorption cross section of 235 GM at 960 nm.2.DPASP demonstrates high mitochondrion-targeting capability and shows better photostability over commercial mitochondrion-targeting probe.With irradiation,Caspase-3 activity of DPASP-stained A549 cells was significantly higher.The simultaneous combination of DPASP and light irradiation will bring concentration-dependent killing effect to A549 cells.3.With two-photon laser scanning,apoptosis of A549 cells can be rapidly activated by DPASP.DPASP-mediated TP-PDT could selectively kill appointed cells in the MTCS but without damage to other cells around.4.DPASP shows splendid tumor retention property after intratumoral injection and its maximum detection depth was up to 1000μm under two-photon fluorescence microscope,which was far better than one-photon imaging.DPASP-induced TP-PDT could effectively inhibit tumor growth of A549 tumor-bearing nude mice.ConclusionIn this study,we designed and synthesized an advanced small-molecule AIE PS,namely DPASP,wihch possesses mitochondrion-targeting capability,excellent photostability,strong two-photon absorption capability as well as high ROS-generation efficiency.DPASP may serve as an excellent two-photon PS and help achieve effective TP-PDT both in vitro and in vivo.The work described here offers a promising strategy to rationally develop AIE two-photon PSs with various specificity and establishes referential experimental program for the research of TP-PDT as well as other stereotactic therapies.