Subcellular imaging and continuous cell tracing of fluorescent probes is significantly important for researchers to investigate the various cell behaviors and processes, such as cell division, cell differentiation, cell cycle, cell apoptosis, etc. [1-5]. During the past decades, a variety of fluorescent agents, including fluorescent proteins, organic small molecular dyes, and quantum dots, have be explored for biological imaging and cell tracing [6-13]. However, some unavoidable drawbacks of fluorescent proteins, such as low photostablity and easy degradation by enzyme, and the small Stokes shift of organic small molecular dyes greatly limit their applications in specific or long-term imaging . In addition, a leakage of the heavy metal components makes the quantum dots highly cytotoxic toward cells in oxidative or extreme environments, which shows less potential in cell imaging utilization [15, 16]. Therefore, it is highly desired that ideal imaging materials with strong fluorescence, large Stokes shift, non-cytotoxicity and high photostability are developed to be applied in biomedical field.
Conjugated polymers, as a class of π-conjugated macromolecules with diverse fluorescence emissions when the backbones are different, have been successfully developed for optical materials in biomedical applications [17-20]. In order to utilize conjugated polymers (CPs) in biological field, researchers make modification to CPs, which leads CPs dispersed in water . Herein, a cationic poly(p-phenylenevinylene) derivative (PPV) containing quaternized N-methyl-imidazole groups as side chains was designed and synthesized. Positively charged PPV could electrostatically absorb onto negatively charged cell surface and further be internalized by endocytosis process. PPV exhibits comparatively high fluorescence yield, large Stokes shift, good biocompatibility and excellent photostablity, and could be used as a lysosome-specific imaging probe and long-term tracing agent.
The synthetic route of PPV is displayed in Scheme 1. Reaction of compound 1 with tributyl(vinyl)tin gives compound 2 in a 64% yield. Compound 1 reacts with compound 2 through Heck coupling to afford compound 3. Then, PPV was obtained by quaternization of the end of side chains with N-methyl-imidazole. The quaternized side chains provide PPV positive charges to make it soluble in water. The optical properties of PPV were investigated in water media. As shown in Fig. S1 (Supporting information), the maximum absorption and emission of PPV are at 453 nm and 555 nm, respectively, and the absolute fluorescence quantum yield of PPV is 4.7%.
The evaluation of biocompatibility toward mammalian cells is of great significance for a promising imaging agent. Thus, the cell viability of MCF-7 treated with PPV was conducted using a standard MTT analysis. As can be seen in Fig. S2 (Supporting information), more than 70% of MCF-7 cells maintain alive when the concentration of PPV is as high as 100 μmol/L, and the cell viability is more than 90% at the concentration applied in the following imaging experiments. Therefore, the newly synthesized PPV has excellent biocompatibility for potential imaging applications.
Because of the excellent fluorescence property and nontoxicity of PPV, the interaction between PPV and MCF-7 cells was subsequently studied. As exhibited in Fig. 1, PPV could successfully enter MCF-7 cells, which confirms that the interaction of positively charged PPV with negatively charged cellular membranes followed by the uptake of cationic PPV by cells. To further explore the distribution of PPV in cells, a commercially lysosome-specific dye (Lyso-Tracker) was utilized to colocalize PPV. The merged image displays that the green fluorescence of PPV and the red fluorescence of Lyso-Tracker overlay very well to provide yellow fluorescence, which indicates that PPV and Lyso-Tracker have a demonstration of good colocalization. Furthermore, the quantitative calculation of Pearson's coefficient by CLSM software based on the merged channels of PPV and Lyso-Tracker fluorescence affords a very high value of 0.98, which implies the primary distribution of PPV in lysosome . The results suggest that PPV could be applied as a promising lysosome-specific imaging material.
|Fig. 1. Confocal images of MCF-7 cells treated with 30 μmol/L PPV costained with 1 μmol/L Lyso-Tracker. The excitation wavelengths of PPV and Lyso-Tracker are 488 nm and 559 nm, respectively.|
In order to investigate whether PPV could stay in cells for a long time to be used as a long-term imaging agent which plays an important role on the study of tumor cell behavior, the photostability and long-term imaging of PPV were evaluated compared with the commercial Lyso-Tracker. As shown in Fig. S3 (Supporting information), the fluorescence of PPV maintains approximately 80% of the initial intensity after 120-second illumination while the fluorescence of Lyso-Tracker remains only 40% of the initial intensity after laser illumination under the same condition, indicative of the good photostability of PPV. Moreover, long-term imaging experiment is performed using PPV and Lyso-Tracker. Fig. 2 displays that MCF-7 cells treated with PPV exhibit obvious green fluorescence after seven-day culture but the red fluorescence of Lyso-Tracker disappears after three-day culture. The absorption and emission spectra of Lyso-Tracker were further measured to compare the optical properties of PPV and Lyso-Tracker (Fig. S4 in Supporting information). Lyso-Tracker exhibits a very small Stokes shift of 19 nm while the Stokes shift of PPV is as large as 102 nm. The larger Stokes shift makes PPV significantly avoid self-absorption during the imaging process. The results implies that PPV is superior to the commercial Lyso-Tracker to be a promising long-term imaging agent.
|Fig. 2. Confocal images of MCF-7 cells treated with 30 μmol/L PPV costained with 1 μmol/L Lyso-Tracker at different culture time. The excitation wavelengths of PPV and Lyso-Tracker are 488nm and 559nm, respectively.|
Additionally, an endocytosis inhibition experiment was conducted to provide direct evidence for the internalization mechanism of PPV in MCF-7 cells. MCF-7 cells were first cultured in different inhibition conditions including low temperature (4 ℃) and treatment with different inhibitors (chlorpromazine (CPZ), dynasore, nystatin and sucrose) at 37 ℃ for one hour in the culture medium without fetal bovine serum (FBS), then the cells were simultaneously incubated with PPV and under different inhibition conditions above for another 6 h. Fluorescence images were taken using confocal laser scanning microscope (CLSM) and quantitatively analyzed fluorescence intensity using the software. As shown in Fig. 3, little green fluorescence of MCF-7 cells incubated with PPV at 4 ℃ and in the presence of dynasore was observed, and MCF-7 cells incubated with PPV and chlorpromazine (CPZ) exhibit weak fluorescence intensity relative to the control group treated with PPV alone, whereas the cells treated with other inhibitors exhibited comparatively as strong fluorescence intensity as the control group. Low temperature (4 ℃) inhibits the energymediated endocytosis process. Dynasore blocks the formation of a pinched-off vesicle and CPZ inhibits the clathrin-related endocytosis. Therefore, the internalization of PPV is an energydependent endocytosis and a pinched-off vesicle formation process because the cell uptake of PPV is significantly inhibited by low temperature and in the presence of dynasore, and the endocytosis of PPV by MCF-7 cells partly depends on the clathrinrelated process because of relatively weak fluorescence observed in the group simultaneously treated with PPV and inhibitor CPZ.
|Fig. 3. Confocal images and normalized fluorescence intensities of MCF-7 cells treated with PPV (30 μmol/L) under various endocytosis inhibition conditions.|
In summary, we have synthesized a cationic PPV derivative with quaternized N-methyl-imidazole groups as side chains. PPV containing positive charges could interact with negatively charged cells and further be internalized by cells through endocytosis route, and PPV has no obvious cytotoxicity toward MCF-7 cells in the experimental concentration range. Specifically, PPV exclusively distributes in lysosomes according to the colocalization assay using a commercial lysosome-specific dye. More importantly, PPV exhibits much larger Stokes shift (102 nm) and higher photostability than the commercial Lyso-Tracker, which implies that PPV could be potential for the alternatives of lysosomespecific agents. Furthermore, long-term imaging study demonstrates that PPV could retain in cells for a relatively long time, which suggests that PPV may be utilized in continuously monitoring cell behaviors including cell division, cell cycle, and cell apoptosis.Acknowledgments
We are grateful to the National Natural Science Foundation of China (Nos. 21504002, 21401008) and Beijing National Laboratory for Molecular Sciences.
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