Study Of Doxorubicin And Curcumin Codelivery Lipid Nanoparticles On Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is a major malignancy worldwide. However, the chemotherapeutic effect of chemotherapeutic agents is often restricted by intrinsic multidrug resistance (MDR) of cancer cells. Doxorubicin (DOX) is one of the most efficacious drugs in the treatment of HCC. However, clinical application of DOX has been severely hindered because of its narrow therapeutic window, critical cardiotoxicity, and the development of MDR. Several studies have reported that curcumin (Cur, a diketone compound from Curcuma longa L.) could be used as a chemosensitizer. But its application is restricted by the poor stability and low bioavailability. In recent years, several studies have reported that codelivery of cytotoxic and chemosensitizing agents in nanoparticles could reverse MDR and enhance cytotoxicity on lung and breast cancer cells. However, these studies are mainly confined to cell experiments, lack of in vivo anti-cancer evaluation and chemosensitive mechanism research.In this thesis, DOX and Cur codelivery lipid nanoparticles (DOX/Cur-NPs) were developed based on the years of research work on lipid nanoparticles in National Engineering Research Center for Nanomedicine to treat HCC. The in vitro cytotoxicity and apopotosis of DOX/Cur-NPs in HCC cells, in vivo anti-HCC efficacy, and probably sensitizing mechanism were studied. The main contents of this thesis are as follows:(1) Cur-loaded lipid nanoparticles (Cur-NPs) were developed and optimized. The composition of Cur-NPs and high pressure homogenization process were screened by solubility and single factor exploration. Cur-NPs were further optimized by a five-level central composite design (CCD).(2) DOX/Cur-NPs were developed from the optimized Cur-NPs. DOX/Cur-NPs were approximately90nm, PDI<0.3, zeta potential<-10mV. The encapsulation efficacy (EE) of DOX and Cur were both>90%. DOX/Cur-NPs exhibited good stability. Differential scanning calorimetry and X-ray diffraction anylasis suggested the amorphous state of DOX and Cur in DOX/Cur-NPs. In vitro release studies indicated sustained release profile of DOX and Cur from DOX/Cur-NPs (2:1,1:1,1:2). The release of DOX fitted Ritger-Peppas model, while the release of Cur fitted Higuchi and Ritger-Peppas models. (3) In vitro cytotoxicity and apoptosis of DOX/Cur-NPs in HCC cells were studied. MTT assay indicated that blank nanoparticles were nontoxic to HepG2cells and L02cells. DOX/Cur-NPs (2:1,1:1,1:2) showed synergistic effects on inbihiting the growth of HepG2cells and recucing the toxicity of DOX in L02cells.1:1might be an optimal weight ratio of DOX and Cur. Annexin V-FITC/PI staining indicated the synergistic effect of DOX/Cur-NPs on promotion of apoptosis in HepG2cells.(4) In vivo anti-HCC efficacy of DOX/Cur-NPs was studied. HCC was induced by diethylnitrosamine (DEN) in male Kunming mice. Evaluation on the changes in body weight, survival rate, liver function, liver/body weight ratio, nodule number in liver surface, maximum nodule size and histopathology indicated the synergistic effect of DOX/Cur-NPs on the growth inbihition of HCC, and the prevention effect of Cur-NPs on the develop of HCC.(5) The probably chemosensitive mechanisms of DOX/Cur-NPs were studied. The mRNA and protein levels of key markers in apoptosis, proliferation, angiogenesis and MDR pathways were determined using real time PCR (RT-PCR), Western blotting and immunohistochemistry. DOX/Cur-NPs improved the therapeutic efficacy on HCC through promoting apoptosis, suppressing proliferation and angiogenesis, and reversing MDR. MTT assay further revealed the reversal effect of DOX/Cur-NPs on MDR. The preventive mechanism of Cur-NPs on HCC was suppressing angiogenesis and hypoxia.Results in this thesis demonstrated that DOX/Cur-NPs syneigistically improved the therapeutic efficacy on primary liver cancer with fine security. The probably mechanism was promoting apoptosis, suppressing proliferation and angiogenesis, and reversing MDR in primary liver cancer. These results would provide experimental evidence for the development of novel codelivery nanoparticles for effective treatment of HCC.