Quantification Of Cellular Uptake And In Vivo Dynamic Cellular Tracking Of Semiconducting Fluorescent Polymer Dots

With the appearance of nanomedicines and the clearer awareness of properties of various cells,cell-based therapy and delivery systems have attracted considerable attention for future medical breakthroughs,owing to their satisfying pharmacokinetic characteristics such as high biocompatibility,long half-life period,significant targeting effect and low toxicity.Several cell-based systems have even shown periodic success in some clinical trials,such as erythrocytes can extent the circulating time of drugs;lymphocytes can improve the targeted safety;stem cells can regenerate damaged tissue and so on.However,although the technology is promising,there are still two key factors to hinder their systemic application in clinic.On the one hand,how to carry out real-time and accurate quantitation of loaded drugs,and on the other hand,how to clearly understand the metabolic process of the transplanted cells in host,these problems must be analyzed by molecular imaging technology in need.Due to the high resolution,non-ionizing radiation,simple operation and multicolor imaging,fluorescence technology present obvious advantages in biological applications such as molecular detection,cell labeling and in vivo imaging.And the guarantee of these performances is closely dependent on the selection and development of fluorescent probes,small molecule dyes are not conducive to long-term monitoring because of their poor photostability;fluorescent protein has potential risks caused by gene transfection;the problems such as heavy metal toxicity of quantum dots,low fluorescence quantum yield of upconversion nanoparticles and weak tissue penetration of carbon dots need to be solved.Polymer dots(Pdots)are widely applied to biosensing,in vivo imaging and phototherapy duo to their large absorption section,high fluorescence quantum yield,superior stability,good biocompatibility and facile surface modification.Aiming at the two problems of cell-based therapy and delivery system mentioned above,the real-time quantification of loaded drugs and the tracking of transplanted cells in host,the results we obtained can be described as below:(1)Measuring cellular uptake of Pdots for quantitative imaging and photodynamic therapy.By comparing with the quantitative results of inductively coupled plasma mass spectrometry(ICP-MS),the same results of intracellular Pdots could be obtained by analyzing the fluorescence signals from fluorescence spectrometry,confocal imaging and flow cytometry.According to our protocol,?1.3×10⁶ Pdots(?20 nm diameter)could be taken up by single MCF-7 cell,and the absolute number of endocytosed Pdots of individual cells could be quantified by comparing its brightness and the average intensity.In addition,the number of endocytosed Pdots could be regulated by the change of labeling time,and based on the photodynamic properties of PFBT@Pt Pdots,their the half maximal inhibitory concentration(IC₅₀)on MCF-7 cells could be obtained under the specific excitation light energy density.(2)Three-dimensional in vivo dynamic cell tracking with doping long-wavelength excitable and near-infrared(NIR)fluorescent Pdots.By doping the deep-red excitation polymer DFDBT and NIR emission dye NIR775 and in virtue of the F(?)rster resonance energy transfer between them,we prepared NIR1 Pdots suitable for in vivo imaging,which possessed strong absorption at 670 nm,fluorescence at 775 nm with quantum yield of 20%.The cell labeling efficiency of Pdots was increased by two orders of magnitude through the mediation of cell-penetrating peptide Tat.In subsequent in vivo experiments,we significantly observed that the fluorescence intensity of the transplanted cells migrated from the lung to the liver from the images in both two-dimensional and three-dimensional imaging.(3)In vivo dynamic tracking of stem cells and cancer cells with blending long-wavelength excitable and NIR fluorescent Pdots.By blending the deep-red excitation polymer DFDBT and NIR emission polymer NIR800 and in virtue of the F(?)rster resonance energy transfer between them,we prepared NIR2 Pdots suitable for in vivo imaging,which possessed strong absorption at 670 nm,fluorescence at800 nm with quantum yield of 22%.Compared with NIR1 Pdots,NIR2 Pdots not only showed a longer wavelength emission,higher quantum yield,but also avoided the risk of the leakage and aggregation of dye.And after the transplantation of the Tat-Pdots labeled cells by tail vein injection,there were obvious distribution difference between stem cells and cancer cells from in vivo imaging.By further fluorescence detection of mice viscera and their frozen sections,the different metabolic trajectories of stem cells and cancer cells in vivo could be analyzed in detail.