br fluorescence signal distribution br Cell experiments
fluorescence signal distribution.
2.7. Cell experiments
In resistant cells, mutant p53 increase the tolerance to chemother-apeutic drug. Depleting mutant p53 would be an efficient way to de-crease drug resistance. In order to prove the ability of BPNs-PDA-PEG-PEITC to suppress the function of mutant p53, western blot assay was used to measure the expression of mutant p53. As shown in Fig. 5a, after incubated with BPNs-PDA-PEG, there were no any change of mutant p53 expression. Compared with the BPNs-PDA-PEG group, it could be found that mutant p53 expression of BPNs-PDA-PEG-PEITC group was remarkably inhibited indicating that the introduction of PEITC could deplete mutant p53 effectively. Besides, the function of BPNs-PDA-PEG-PEITC/DOX to inhibit the expression of mutant p53 in MCF-7/ADR was also estimated. As shown in Fig. S11, the inhibition mutant p53 expression of BPNs-PDA-PEG-PEITC/DOX group was re-markable compared with the BPNs-PDA-PEG/DOX group indicating that BPNs-PDA-PEG-PEITC/DOX still can inhibit mutant p53 expres-sion. In vitro cell cytotoxicity tests (Fig. 5b and c) toward MCF-7 cells, and a drug resistant one, MCF-7/ADR cells, were used to estimate the therapeutic effect of different samples after reversing resistance. It could be found that BPNs-PDA-PEG and BPNs-PDA-PEG-PEITC had negligible cytotoxicity on MCF-7 and MCF-7/ADR KPT-185 with con-centration increase, suggesting the good biocompatibility both BPNs-PDA-PEG and BPNs-PDA-PEG-PEITC. Besides, free DOX and DOX-loaded nanosheets had obvious dose-dependent cytotoxic effect on MCF-7 cells. Free DOX and BPNs-PDA-PEG/DOX had a mild
cytotoxicity on MCF-7/ADR cells due to the strong drug resistance. However, when the MCF-7/ADR cells were treated with BPNs-PDA-PEG-PEITC/DOX, a reduced cell survival rate was observed as the concentration of nanocomposites rose. These results suggest that the nanocomposites modified by PEITC could reverse the MDR of MCF-7/ ADR cells and enhance drug therapeutic efficiency. In addition, in consideration of PTT and PDT could also induce an antitumor effect, thus in vitro cell cytotoxicity assay was also used to evaluate synergistic PTT/PDT/chemo-therapy efficiency. As could be seen in Fig. 5c, BPNs-PDA-PEG-PEITC/DOX, BPNs-PDA-PEG-PEITC + 660 nm, BPNs-PDA-PEG-PEITC + 808 nm displayed concentration-dependent toxicities on MCF-7/ADR. When MCF-7/ADR treated with the combined dual-treatment, a more effective cancer treatment efficiency could be ob-served. The group of BPNs-PDA-PEITC/DOX + 660 nm + 808 nm pre-sented the lowest cell viability demonstrating the excellent antitumous effect of synergistic PTT/PDT/chemo-therapy. As shown in Fig. 5d, the BPNs-PDA-PEG-PEITC/DOX could be internalized by MCF-7/ADR cell through endocytosis and PEITC could decrease the mutant p53 level realizing the reversal of drug resistance.
The behaviors of BPNs-PDA-PEG-PEITC/DOX in animals were in-vestigated via utilizing in vivo MRI. According to previous literature, Mn2+, Zn2+, Cu2+ and Fe3+ ions could be chelated with the phenolic hydroxyl group . Among these metal ions, Mn2+ could be utilized as MRI contrast agent. In this theranostic nanoplatform, Mn2+ was in-tegrated with phenolic hydroxyl groups of PDA layers successfully via
Fig. 4. (a) UV–visible absorption of the BPNs-PDA-PEG-PEITC, DOX, and BPNs-PDA-PEG-PEITC/DOX. (b) Release profiles of drug from the BPNs-PDA-PEG-PEITC/ DOX dispersed in pH 7.4, 6.8 and 5.0 buffer at 37 °C. (c) The NIR irradiation-responsive release profiles of drug from the BPNs-PDA-PEG-PEITC/DOX dispersed in pH 7.4, 6.8 and 5.0 buffer. Confocal fluorescence images of MCF-7/ADR cells incubated with BPNs-PDA-PEG-PEITC/DOX for 2 h (d) without and (e) with NIR laser irradiation (1.2 W/cm2) and cell fluorescence intensity distribution analysis for the yellow square marked area. Red-fluorescence from DOX and blue-fluorescence from DAPI. The scale bar: 50 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
chelation. As shown in Fig. 6a and b, the obtained BPNs-PDA-PEG-PEITC-Mn/DOX presented obvious brightening effect with the solution concentration increase under T1-weighted MRI. The T1 relaxivity (r1) value of BPNs-PDA-PEG-PEITC-Mn/DOX was calculated to be 9.66 mM−1 s−1 indicating potential capability for T1-weighted MRI. Afterwards, in vivo experiment was performed to assess the T1 weighted MRI capability of BPNs-PDA-PEG-PEITC-Mn/DOX (Fig. 6c). The tumor-bearing mice were intravenously injected with BPNs-PDA-PEG-PEITC-Mn/DOX (10 mg/kg, 100 μL) for MRI. Compared with pre-injection, at 1 h post-injection the tumor region presented a brightening effect. Furthermore, the stronger T1-weighted MRI signal could be observed in the tumor region at 6 and 12 h post-injection and a software named ImageJ was used to measure the T1 MRI signal intensities of tumor region (Fig. S12) which further verify the enhancement of signal in-tensity in the tumor region after injection of BPNs-PDA-PEG-PEITC-Mn/ DOX. These results indicated the effective tumor accumulation of BPNs-PDA-PEG-PEITC-Mn/DOX via EPR effect and blood circulation.