Preparation And Dual Emissive Imaging Application In Cancer Cells Of Stimuli-Responsive Polymer Dots

In recent years,polymer dots(Pdots)have been developed as a new organic fluorescent nanoparticles based on polymers.Polymer quantum dots have many advantages such as: excellent optical properties,diverse structures,easy surface modification and good biocompatibility.Therefore,they have important application potential in biological imaging,sensing and detection,drug delivery and therapeutic diagnosis.However,most of the Pdots what are currently reported are conventional semiconducting polymer quantum dots,which are prone to ?-? stacking in the aggregated state,resulting in fluorescence quenching(aggregation-caused quenching,ACQ)due to the large conjugated structure.This situation limits its bioimaging applications in aggregated or high concentrations.If the "Aggregation-induced emission(AIE)" property is introduced into the Pdots material,the ACQ problem can be fundamentally solved.In addition,intelligent amphiphilic polymers are widely used in biomedicine,nanotechnology and other fields because of their unique self-assembly ability and drug loading behavior.Based on the existing research of the research group,this paper combines the advantages of AIE,dual fluorescence and intelligent response to design and prepare five non-semiconductor Pdots fluorescent nanomaterials with different fluorescence characteristics.Moreover,the effects of Pdots structure on its self-assembly behavior,fluorescence performance and cell imaging were systematically studied.Based on this,the double-fluorescent Pdots with the best optimization performance were selected.The main research contents are as follows:1.Preparation and Dual Emissive Imaging Application in Cancer Cells of Temperature Responsive Polymer DotsThe first part of the work designed and synthesized single,double,four-arms AIE molecules containing a free radical initiator: TPE-PCL-azo-initiator.Then,the in-situ initiation polymerization method is used to realize the covalent bonding of the temperature sensitive polymer poly(N-isopropylacrylamide)(PNIPAM)with the AIE molecule.Simultaneous effectively coordinate with the rare earth element Eu(III)formed single,double,four-arms amphiphilic polymers with dual fluorescence emission TPE-PCL-PNIPAM-Eu(III),2s-TPE-PCL-PNIPAM-Eu(III),4s-TPE-PCL-PNIPAM-Eu(III).Finally,three kinds of AIE type Pdots were prepared by nanoprecipitation: TPE-PCL-b-PNIPAM-Eu(?),2s-TPE-PCL-b-PNIPAM-Eu(?),4s-TPE-PCL-b-PNIPAM-Eu(?).The structure and composition of the synthesized polymers were characterized by NMR,GPC,IR,XPS;The morphology and particle size of the three Pdots were investigated by TEM and DLS.The results show that the three Pdots are regular small spheres,especially the four-arms Pdots have the smallest particle size about 2.3 nm;The optical properties of the three Pdots were studied by fluorescence spectroscopy.The results show that the four-arms Pdots have superior AIE properties,dual fluorescence properties and sensitivity to temperature,which compared to single-arm and double-arms Pdots.The four-arms Pdots is an excellent temperature-sensitive dual-fluorescence nanomaterial because its LCST is about 37.5 °C,which is close to human body temperature;The bioavailability of three Pdots was investigated by cell imaging experiments.The results showed that all three Pdots have low toxicity and good biocompatibility.By comparing and analyzing the staining effects of three different types of cancer cells(Hep G2,A549,He La cells),it was found that four-arms Pdots were more easily captured by cancer cells,and two-color(blue,red)fluorescence imaging in cancer cells was achieved by regulated excitation.This can effectively eliminate the interference of biological tissue autofluorescence.2.Preparation and Dual Emissive Imaging Application in Cancer Cells of p H-Responsive Polymer DotsThe second part of the work is the ring-opening polymerization of AIE molecule TPE-4OH with caprolactone(?-CL)to obtain polymer 4s-TPE-PCL.The macroinitiator 4s-TPE-PCL-AZO was further synthesized by DCC condensation reaction with ACVA.Then,the p H-sensitive monomer acrylic acid(AA)is in-situ initiated to obtain the p H-responsive amphiphilic copolymer 4s-TPE-PCL-b-PAA,and then coordinated with Eu3+ to obtain an adjustable double fluorescent molecule 4s-TPE-PCL-b-PAA-Eu(III).Finally,p H-sensitive Pdots with a particle size about 2.8 nm were prepared by nanoprecipitation.The structure and composition of the polymers were characterized by NMR,GPC,IR,XPS;The dual fluorescence properties,AIE properties and p H responsiveness of Pdots were studied by fluorescence spectroscopy.The results show that the Pdots have two fluorescence emission centers and the fluorescence color is adjustable,and the blue light is sensitive to p H;In addition,by imaging evaluation of Pdots in Hep G2,A549,He La cells,it was found that the Pdots have good biocompatibility,especially for He La cells.3.Preparation and Dual Emissive Imaging Application in Cancer Cells of Temperature / p H dual-response type Responsive Polymer DotsIn the third part,the amphiphilic copolymer with AIE characteristics was prepared by using TPE-4OH as the luminophore,PCL as the hydrophobic part and temperature/p H double-responsive polymer PVP as the hydrophilic part.Then introducing the rare earth element Eu(III)for coordination,the temperature / p H dual response type Pdots 4s-TPE-PCL-b-PVP-Eu(III)was obtained by coprecipitation.The structure and composition of the polymers were characterized by NMR,GPC,IR,XPS;The morphology and particle size of Pdots self-assembly were investigated by TEM and DLS,indicating that the Pdots are a nanosphere with a particle size about 2.6 nm;The dual fluorescence properties,AIE properties,temperature sensitivity and p H sensitivity of Pdots were studied by fluorescence spectroscopy.It was found that 4s-TPE-PCL-b-PVP-Eu(III)Pdots have excellent optical properties,the fluorescence color and intensity can be tune.Pdots are responsive to T and p H with an LCST of approximately 37 °C.In addition,the effect of two-color fluorescence imaging in different types of cancer cells was studied by adjusting the excitation wavelength,and it was found that the Pdots had excellent imaging effects in HepG2 cytoplasm.