Multi-walled Carbon Nanotubes Drug Delivery System In Improving The Effect Of Antioxidants

The maintenance of normal pregnancy depends on normal placental function. A large number of studies have shown that oxidative stress of utero-placental tissue especially trophoblasts plays a pivotal role in the onset and development of many placental-related disorders, such as preeclampsia, fetal distress, fetal growth restriction, abortion etc. Therefore, therapeutic effect of antioxidants arouses more and more attention. There are mainly two kinds of antioxidants:antioxidant enzymes and non-enzymatic antioxidants, both of which have shortcomings. The former are not easy to enter cells, the latter do harm to placenta when in high doses, so building a drug delivery system which can easily penetrate cells with controlled drug release to enhance the curative effect of antioxidants is necessary. Carbon nanotubes (CNTs), formed by carbon atoms, have structures of one-dimensional hollow tubular bodies. Since CNTs display unique structures and remarkable physical properties, various applications of CNTs have emerged in materials, catalysis and life sciences etc, especially in the matter of drug delivery. In this research, we first investigated the oxidative damage of the cell line HTR-8/SVneo treated with different concentrations of hydrogen peroxide (H2O2) for different time, to establish the oxidative stress model of human trophoblast. Secondly, we prepared carboxyl modified MWNTs (MWNT-COOH) and SOD covalently functionalized MWNTs (SOD-g-MWNT), detected their features and studied their biological toxicity. Finally, on the basis of the above-mentioned oxidative stress model, we compared the different effect of GSH, MWNT-COOH+GSH and SOD-g-MWNT+GSH on attenuating cell oxidative damage, aimed at searching a new method not only to improve antioxidants’curative effect, but simultaneously avoid their side effects.Section1Establishment of hydrogen peroxide induced human trophoblast HTR-8/SVneo oxidative stress model.Objective:Study the impact of hydrogen peroxide (H2O2) on the cell line HTR-8/SVneo, and establish the oxidative stress model of human trophoblast.Study design:HTR-8/SVneo were treated with different concentrations of H2O2for different time, then cell proliferation was measured by CCK-8, the activity of superoxide dismutase(SOD) in cell culture supernatant and lactate dehydrogenase(LDH) leakage were detected by spectrophotometry, intracellular superoxide anion and apoptosis rate were detected by the method flow cytometry analysis. Results:Overall, H2O2caused the proliferation of HTR-8/SVneo inhibited, the activity of SOD decreased and LDH leakage increased, also promoted cell apoptosis and intracellular superoxide anion production with concentration and time-dependent. However, this study also found that when treated with125μM H2O2, the above-mentioned effect can be totally opposite.Conclusion:When treated with H2O2at low concentrations, the intracellular oxidation and antioxidation balance in HTR-8/SVneo gradually tend to the side of the antioxidation. With the concentration of H2O2as well as the role of time increased, the oxidative damage in HTR-8/SVneo tends to be significantly. According to this research, the appropriate condition of establishing the oxidative stress model of HTR-8/SVneo is treated with250uM H2O2for4hours. The effect of oxidative stress and lipid peroxidation on cell proliferation and apoptosis was diphasic, in which extracellular signal-transduction pathway may play an important regulatory role. The specific mechanism needs further research.Section2Study on the preparation and biological safety of carboxyl modified MWNTs and SOD covalently functionalized MWNTs.Objective:Prepare carboxyl modified MWNTs (MWNT-COOH) and SOD covalently functionalized MWNTs (SOD-g-MWNT), and detect their features, study their biological toxicity.Study design:Carboxyl modified MWNTs were obtained by oxidation of pristine MWNTs, and SOD covalently functionalized MWNTs were synthesized via amidation reaction. Transmission electron microscopy and fourier transform infrared spectrum were used to characterize MWNT-COOH and SOD-g-MWNT. The grafted ratio of SOD was evaluated by TGA and the activity of SOD was test by spectrophotometry. Furthermore, SOD and SOD-g-MWNT were labeled by FITC to trace the delivery of SOD by MWNTs into cells, using flow cytometry analysis. Finally, the toxicity of MWNT-COOH and SOD-g-MWNT was also studied through CCK-8method.Results:After modified by carboxyl, the length of MWNTs was greatly shortened, and could well disperse in aqueous solution. Via amidation reaction, SOD was successfully covalently connected to MWNT-COOH, and the grafted ratio of SOD evaluated by TGA was11wt%, without apparent destruction of the activity of SOD. Traced by FITC, we could see that SOD was delivered by MWNTs into cells easily. The fluorescent positive rate tested by flow cytometry analysis was68.57%, much higher than FITC-SOD treated group. In24hours, both MWNT-COOH and SOD-g-MWNT had no significant impact on cell proliferation.Conclusion:Carboxyl functionalization is an effective way to improve the biocompatibility and cell penetrability of MWNT. SOD-g-MWNT can improve cell uptake rate of SOD without doing damage to the activity of SOD. Both MWNT-COOH and SOD-g-MWNT show no significant cell toxicity.Section3Impact made on the antioxidant effect of GSH by MWNT-COOH and SOD-g-MWNT drug delivery system.Objective:On the basis of the above-mentioned oxidative stress model, we compared the different effect of GSH, MWNT-COOH+GSH and SOD-g-MWNT+GSH on attenuating cell oxidative damage, aimed at searching a new method not only to improve antioxidants’ curative effect, but simultaneously avoid their side effects.Study design:First, MWNT-COOH and SOD-g-MWNT were used as drug carriers to adsorb GSH, then GSH, MWNT-COOH+GSH and SOD-g-MWNT+GSH were separately used to treat above-mentioned HTR-8/SVneo oxidative stress model. We used CCK-8cell proliferation test to evaluate the effect of these three methods on cell viability, flow cytometry analysis detecting intracellular ROS to compare their effect on attenuating cell oxidative damage.Results:Compared with GSH, MWNT-COOH+GSH could better increase cell viability as well as reduce the generation of intracellular ROS. However, the effect of SOD-g-MWNT+GSH was not that good, even inferior to GSH in reducing intracellular ROS.Conclusion:The therapeutic effect of MWNT-COOH+GSH was better than GSH, which means that carried by MWNT-COOH, the antioxidant effect of GSH was improved. The specific mechanism needs to be further studied. But we think that MWNT-COOH is expected to be an excellent carrier of small molecular antioxidants. The reason why SOD-g-MWNT instead reduces the antioxidant effect of GSH needs to be further defined.