| Cells are the building blocks of life. It’s significant to study the constituents within cells to reveal the principles of life activities. Most traditional researches are performed using bulk methods based on average events of a great many cells. However, heterogeneity is an inherent character of cell populations. The average results could not reflect the real structure of the cell populations and often provide misleading results. On the other hand, single-cell analysis (SCA) could examine the variation within cells to get the real information. Besides, it is important to study the cellular behaviors of drug uptake to analyze the pharmacokinetics, pharmaco dynamics and mechanisms of drug action. Based on these, in this dissertation, we constructed two single-cell analysis systems of capillary electrophoresis coupled with laser-induced fluorescence detection (CE-LIF) and microfluidic chip coupled with laser-induced fluorescence detection (Microfluidics-LIF). The cellular uptake of doxorubicin (DOX) in single cancer cells were analyzed using CE-LIF and Microfluidics-LIF methods. The results showed that marked heterogeneity exists within DOX uptake by cells. This drug uptake variation leads to important biological effects. Adjusting the drug schedules or inhibiting the function of cellular surface P-gp could lower the cellular heterogeneity in drug uptake, and thus improve the anticancer outcome. The main works are as below:(1) The home-built CE-LIF analytical system with the orthogonal optical design was constructed. The limit of detection (LOD, S/N=3) of fluorescein sodium reached2.74×10-12M (1.03×10-12g/mL) under the optimal conditions.(2) The CE-LIF analytical method for DOX was set up. The CE running buffer was optimized as10mM Borate-SDS solution. The LOD (S/N=3) of concentration and quantity of DOX reached1.55×10-10M (0.084ng/mL) and56.1zmol, respectively The concentration linear range was between2×10-8and1×10-5M, and the quantity linear range was between7.3to3630amol with the correlation coefficient r=0.9979. The CE-LIF method is suited to analyze the DOX uptake within a one cell with high sensitivity, good linear correlation and wide quantitative range.(3) The DOX uptake in single human leukemia K562cells treated with different concentration of DOX for different period was detected using CE-LIF. An obvious heterogeneity in DOX uptake among single cells was observed. The RSD%of DOX uptake amounts ranged from24.0%to61.1%. The cellular heterogeneity in DOX uptake decreased with increasing drug concentration, and reached to equilibrium, but it did not exhibit regular changes with increasing drug exposure time. The mean DOX uptake showed a good linear function of drug concentration (r≥0.9667), and its correlation with drug exposure time presented the real changing process of DOX within cells.(4) To investigate the biological consequences of the cellular heterogeneity in drug uptake, the cell-to-cell difference in DOX uptake by single K562and MCF-7cells treated with different schedules of DOX (Single-high dose and multi-low dose schedules) was analyzed using CE-LIF. The corresponding anti-cancer effects were also examined. The results showed that marked heterogeneity lies within cell populations. The single-high dose schedules of DOX resulted in lower cellular heterogeneity in DOX uptake, higher mean DOX uptake and superior cytotoxicity of DOX to cancer cells in comparison with the multi-low dose schedules of DOX. The addition of verapamil (VER) as a P-glycoprotein (P-gp) inhibitor effectively reduced the cellular heterogeneity in DOX uptake, implying that the variation in DOX uptake by cells correlates with the cellular membrane surface P-gp. The anti-cancer outcome could be improved by adjusting the drug schedules or inhibiting the function of cellular surface P-gp.(5) To improve the throughput of single-cell analysis using CE, the analytical method using microfluidics was also investigated. The microfluidic chip electrophoresis (MCE)-LIF/CE-LIF analytical system was constructed with the small incident angle optical design. The LOD of fluorescein sodium reached6.0×10-11M under the optimal conditions, being1%of LOD provided by the commercial instruments. The LOD (S/N=3) of concentration and quantity of DOX reached4.3×10-10M and105.4zmol, respectively, which is sufficient to detect DOX uptake in a single cell.The analytical method of DOX using MCE-LIF was established. The DOX was injected into the home-made cross PDMS chip by pinched mode and was analyzed using MCE-LIF. The LOD (S/N=3) of DOX reached2.86×10-10M under the optimal conditions. The linear range was1.0×10-8-2.5×10-7g/mL with the correlation coefficient r=0.9580. The MCE-LIF method could be used to detect DOX uptake in a single cell.(6) Continuous single-cell analysis method using microfluidic chips was set up. Single cells were continuously induced into the newly-designed PDMS chip by means of laminar flow in the micro-channel; the fast single-cell lysis was realized by mixing of laminar flow, which was achieved with the optimization of focus length, channel angle, symmetry and micro-structures in the channel. The throughput of single-cell analysis reached300cells per hour that is100times faster than the CE method. The DOX uptake and expression of membrane surface P-gp of single cells were detected using microfluidics. The results indicated that the cellular heterogeneity in DOX uptake is lower for the cells treated with single-high dose schedules of DOX than that for the cells treated with multi-low dose schedules of DOX. The addition of VER could reduce the cell-to-cell variation in DOX uptake. Similarly, the cellular heterogeneity in surface P-gp expression for the cells treated with single-high dose DOX schedules is lower than that for the cells treated with multi-low dose DOX schedules. The addition of VER could reduce the cellular heterogeneity in surface P-gp expression. These results suggested that the cellular heterogeneity in DOX uptake is correlated with the cellular heterogeneity in surface P-gp expression and the function of surface P-gp. |
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