刺激响应型纳米探针的制备及其对卵巢癌细胞的磁共振成像与药物输运研究

目的  构建基于肿瘤微环境下刺激响应型纳米探针介孔二氧化硅@阿霉素@二氧化锰纳米片(mSiO₂@DOX@MnO₂)，探索其在卵巢癌细胞的特异性MRI与药物释放性能。方法  以转铁蛋白为稳定剂和靶向分子，通过超声分散法制备MnO₂纳米片，以MnO₂纳米片为门控，通过静电相互作用构建mSiO₂@DOX@MnO₂纳米探针。检测其Zeta电位、微观形貌、药物控释、光学性质及MRI性能。采用细胞增殖/毒性检测试剂盒检测纳米复合物对HO-8910卵巢癌细胞和CHO仓鼠卵巢细胞的细胞毒性。检验其在肿瘤细胞内的药物释放情况及细胞水平的MRI成像效果。结果  mSiO₂@DOX@MnO₂纳米探针在正常生理环境下几乎无MR信号，药物释放率小于10%。在肿瘤微环境高谷胱甘肽（GSH）浓度下产生较强的T1加权信号，其T1弛豫效率r1为5.86 mmol/（L·s）。在pH=5.0的酸性环境中，DOX的释放率在15 h左右开始维持在相对稳定水平，释放率约为33%。相同pH=7.4条件下，GSH高浓度组较低浓度组释放率明显增高，约为55%。当pH=5.0时，GSH浓度为10 mmol/L时，释放率最高，高达80%。mSiO₂@DOX@MnO₂纳米探针在实验浓度内对肿瘤细胞毒性较强，对正常CHO细胞毒性较弱，当mSiO₂@DOX@MnO₂纳米复合物中DOX含量约为100 μg/mL，HO-8910细胞存活率仅为24%，CHO细胞的存活率约为86%。HO-8910细胞、CHO细胞在不同质量浓度mSiO₂@DOX@MnO₂作用下存活率差异均有统计学意义（P < 0.05）。当锰离子浓度为1.12 mmol/L时，HO-8910细胞组的T1驰豫时间为467.60±4.45 ms，CHO细胞组的T1驰豫时间为1681.47±1.88 ms。同一浓度下的纳米探针对HO-8910细胞和CHO细胞MRI的T1弛豫时间两两比较差异均有统计学意义（P < 0.05）。结论  构建的mSiO₂@DOX@MnO₂纳米探针可靶向识别卵巢癌细胞，在肿瘤细胞酸性环境下及高水平GSH刺激响应下实现T1加权成像、药物的精准释放，可实现卵巢癌细胞水平的靶向磁共振成像及治疗。

Development of stimuli-responsive nanoprobes with the aim of enabling targeted magnetic resonance imaging and drug delivery for ovarian cancer cells

Objective To fabricate nanoprobes consisting of tumor microenvironment stimuli-responsive mesoporous silicon dioxide@doxorubicin@manganese dioxide (mSiO2@DOX@MnO2) and assess their efficacy in terms of MRI and drug release in ovarian cancer cells. Methods The preparation of MnO2 involved ultrasonic disperion with transferrin serving as both a stabilizer and a targeted agent. The mSiO2@DOX@MnO2 nanoprobes were synthesized through electrostatic interaction utilizing MnO2 nanosheets as a gate. The nanoprobes were subsequently characterized for zeta potential, morphology, drug release kinetics, optical properties, and MRI properties. The cytotoxicity of the nanocomplexes was assessed against HO-8910 ovarian cancer cells and CHO hamster ovarian cells using the cell counting Kit-8 (CCK-8) assay. The drug release within tumor cells was investigated using a confocal laser scanning microscope. Furthermore, the MRI imaging effect of the nanoprobes on HO-8910 cells was evaluated. Statistical analysis was performed using one-way analysis of variance and the LSD test. Results The mSiO2@DOX@MnO2 nanoprobes exhibited minimal magnetic resonance signal and a low drug release rate (less than 10%) in a normal physiological microenvironment. However, in the presence of glutathione (GSH), a strong T1 weighted signal was obseved, with a T1 relaxation efficiency of 5.86 mmol/(L·s). It could be observed that in an acidic environment with a pH of 5.0, the release rate of DOX reaches a relatively stable level at approximately 15 h, maintaining around 33%. Under the same condition with a pH of 7.4, the high concentration group exhibits significantly higher GSH release rate compared to the low concentration group, reaching about 55%. The highest release rate is achieved when both pH and GSH concentration are set at 5.0 and 10 mmol/L respectively, reaching up to 80%. The mSiO2@DOX@MnO2 nanoprobe demonstrates potent toxicity against tumor cells while exhibiting minimal toxicity towards normal CHO cells at experimental concentrations. When the DOX content in the mSiO2@DOX@MnO2 nanocomplex was about 100 μg/mL, HO-8910 cell survival rate is only about 24%, whereas CHO cell survival rate remains around 86%. Cell cytotoxicity tests demonstrated significant differences in the survival rates of HO-8910 cells and CHO cells when treated with varying concentrations of mSiO2@DOX@MnO2 (P < 0.05). When the concentration of manganese ions was 1.12 mmol/L, the T1 relaxation time of the HO-8910 cell group was 467.60±4.45 ms, while that of the CHO cell group was 1681.47±1.88 ms. The T1 values of the HO-8910 cells and CHO cells groups showed significant differences when treated with equivalent concentrations of mSiO2@DOX@MnO2 (P < 0.05). Conclusion The developed mSiO2@DOX@MnO2 nanoprobe exhibits targeted identification and localization capabilities towards ovarian cancer cells, enabling T1-weighted imaging and precise drug release in acidic tumor microenvironments with high levels of GSH stimulation response, thereby facilitating targeted magnetic resonance imaging and treatment at the cellular level for ovarian cancer.