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Abstract
Background:
Cisplatin (CDDP), a widely used chemotherapeutic agent against hepatocellular carcinoma (HCC), faces severe resistance and hepatotoxicity problems which can be alleviated through combination therapy.
Purpose:
The objective of this study was to develop a pH-dependent calcium carbonate nano-delivery system for the combination therapy of CDDP with oleanolic acid (OA).
Methods:
A microemulsion method was employed to generate lipid coated cisplatin/oleanolic acid calcium carbonate nanoparticles (CDDP/OA-LCC NPs), and the loading concentration of CDDP and OA was measured by atomic absorption spectroscopy and HPLC respectively.Transmission electron microscopy (TEM) was used to examine the nanoparticles morphology while its pH dependent release characteristics were investigated through in vitro release study. Cellular uptake was examined through a fluorescence microscopy. Apoptotic assays and western blot analysis were conducted to explore the synergistic apoptotic effect of OA on CDDP against HCC cells. The hepatoprotective of OA for CDDP was evaluated through H&E staining.
Results:
TEM analysis revealed nanoparticles spherical shape with an average particle size of 206±15 nm, and the overall entrapment efficiency was 63.70%±3.9%. In vitro drug release study confirmed the pH-dependent property of the formulation, with the maximum CDDP release of 70%±4.6% at pH 5.5, in contrast to 28%±4.1% CDDP release at pH 7.4. Annexin V-FITC/PI assay and cell cycle analysis confirmed that CDDP and OA synergistically promoted greater HepG2 cells apoptosis for the CDDP/OA-LCC NPs as compared to their individual free drug solutions and NPs-treated groups. Western blot analysis also proved that CDDP/OA-LCC NPs induced the apoptosis by enhancing the proapoptotic protein expressions through downregulating P13K/AKT/mTOR pathway and upregulating p53 proapoptotic pathway. OA helped CDDP to overcome the resistance by downregulating the expression of proteins like XIAP, Bcl-2 via NF-κB pathway. OA also significantly alleviated CDDP-induced hepatotoxicity as evident from the decreased alanine transaminase, aspartate transaminase levels and histochemical evaluation. The possible mechanism may be related to the Nrf-2 induction via its antioxidant mechanism to maintain the redox balance and reduction in CYP2E1 activity which can lead to ROS-mediated oxidative stress.
Conclusion:
These results suggest that CDDP/OA-LCC NPs have promising applications for co-delivering CDDP and OA to synergize their anti-tumor activity against HCC and to utilize OA’s protective effect against CDDP-induced hepatotoxicity.
Acknowledgments
This research was supported by National Natural Science Foundation of China (NSFC) (No. 81673368), the Fundamental Research Fund for the Central Universities of Huazhong University of Science and Technology (HUST) (2016YXMS140, 2016YXMS147, 2016JCTD109, and 2014TS090).
Disclosure
The authors report no conflicts of interest in this work.
Supplementary materials
Figure S1 Cytotoxicity assay of free CDDP and free OA against HepG2 cells. (A) MTT assay results of free CDDP solution against HepG2 cells for 24, 48, and 72 h; (B) MTT assay results of free OA solution against HepG2 cells for 24, 48, and 72 h. Data presented as mean±SD (n=5).
Abbreviations: CDDP, cisplatin; OA, oleanolic acid.
Figure S2 CDDP/OA-LCC NPs induces apoptosis in HepG2 cells. Hoechst 33,258 nuclear staining cell apoptosis images. (A) Control; (B) CDDP-Sol; (C) CDDP-LCC NPs; (D) OA-Sol; (E) OA-LCC NPs; (F) CDDP/OA-LCC NPs.
Abbreviations: CDDP, cisplatin; OA, oleanolic acid; LCC, lipid coated calcium carbonate; NP, nanoparticles.
Figure S3 Graphical representation depicting the proposed protective mechanisms of oleanolic acid (OA) co-administration on liver injury induced by cisplatin
