| Hepatocellular carcinoma (HCC) is the fifth most common cancer and it remains the third most frequent cause of cancer-related death worldwide. The incidence of HCC is still increasing in recent years. China is one of the most common countries for HCC and more than half of all global cases occur in China alone. HCC presents a significant economic and societal burden in China.At present, the methods for the treatment of HCC include surgery such as curative resection and liver transplantation, radiotherapy, local treatment such as transarterial chemoembolization and radioembolization, chemotherapy and so on. Sorafenib (SF) was the first targeting chemotherapeutic approved for HCC. On the one hand, it shows remarkable inhibition of RAF/MEK/ERK signal transduction pathway, thus it directly induces inhibitory effects during cell proliferation directly. On the other hand, it induces inhibitory effects during tumor angiogenesis by suppressing the vascular endothelial growth factor (VEGF) receptors, and platelet-derived growth factor (PDGF) receptor. SF shows good antitumor effect because of the two action pathways, thus it is commonly used as a first-line treatment for advanced HCC in clinical applications. However, SF shows some disadvantages such as poor solubility in water, low oral bioavailability (approximately 8.43％), high interindividual variability in terms of its pharmacokinetics and multiple human dose-limiting toxicities including anorexia, gastrointestinal bleeding, hypertension and skin toxicity which greatly restricts its therapeutic efficacy against cancer. Thus, it has been one of the most important scientific issues to improve the HCC treatment effect of SF by choosing appropriate methods.Combination therapy is one of the most important methods to improve the tumor treatment effect in clinical practice. It can be used in order to enhance therapeutic efficacy, decrease systemic toxicity and overcome drug resistance and so on. In the tumor combination therapy, the commonly used combinations are drug and drug combination, drug and gene combination, drugs and tracer combination and drugs and sensitization agent combination, etc.In this work, SF and gadolinium (Gd) co-loaded liposomes (SF/Gd-liposomes) and SF and ceramide (CE) co-loaded liposomes (SF/CE-liposomes), were constructed according to the principle of combination therapy. SF was served as the therapeutic agent model and liposome was chosen as the basic nanocarriers. The two co-loaded liposomes were studied on different aspects, drugs and MRI tracer combination and drugs and sensitization agent combination, respectively. The two co-loaded liposomes were evaluated in terms of pharmaceutical characteristics in vitro and vivo, respectively. The main methods and results were as follows:1. Determination of drug content and encapsulation efficiency of the co-loaded liposomesThe drug content and encapsulation efficiency of the co-loaded liposomes were determined by HPLC method. The centrifugalization and membrane filtration were applied to separate free SF.2. Preliminary Studies on SF/Gd co-loaded liposomes (SF/Gd-liposomes)The SF/Gd-liposomes were prepared by thin film hydration method. Malvern Zetasizer Nano ZS instrument was used to determine the mean diameters, particle size distributions and zeta potentials of the liposomes. The morphology of the liposomes was characterized by Transmission electronic microscopy. The Gd content of the SF/Gd-liposome was determined by inductively coupled plasma optical emission spectroscopy (ICP-OES). HPLC method was used to determine the encapsulation efficiency and drug loading of the co-loaded liposomes. The release behavior was studied in vitro with a dialysis method. The size changes at 4 ℃ and 25℃ for 10 d were used to study the preliminary stability of SF/Gd-liposomes. The in vitro cytotoxicity of SF/Gd-liposomes on HepG2 cells was evaluated using MTT assay. The MRI imaging abilities of the SF/Gd-liposomes were investigated by magnetic resonance instrument in vitro and in vivo. In vivo anti-tumor effects were then studied on H22 tumor-bearing mice. Furthermore, the in vivo preliminary safety of SF/Gd-liposomes was tested by hemolysis assay and histological examination of main organs.The morphology of SF/Gd-liposomes was spherical or ellipsoidal shapes and the mean particle size and PDI, zeta potential, encapsulation efficiency and drug loading were(180±7) nm and 0.20, (-7±1)mV, (96±2)％and (4.3±0.1)％, respectively. The concentration of Gd was 181±5 mg/L. No apparent changes of the liposome sizes were observed in 10 d both at 4 ℃ and 25℃ which indicated that the liposomes could remain intact in this condition. The SF/Gd-liposomes exhibited the slow and sustained release kinetics of SF and only 50％of the SF was released at approximately 60 h. The release of Gd was faster and completed. At 12 h, the release of Gd could reach 100%. This was benefit for achieving therapy and non-invasive imaging in the meantime. In the in vitro cytotoxicity test, SF/Gd-liposome showed lower cell cytotoxicity in comparison with the SF solution (p<0.05). Slightly lower imaing ability was shown by SF/Gd-liposomes compared to Magnevist(?) in the in vitro MRI test. The relaxivity of SF/Gd-liposomes and Magnevist(?) was 3.2 mM-1s-1 and 4.5 mM-1s-1, respectively. While in the in vivo MRI test, SF/Gd-liposome had significant target enhancements in tumor, liver, heart and muscle tissues in comparison with Magnevist(?). SF/Gd-liposome showed longer imaging times and higher signal enhancement especially in the tumor tissue. SF/Gd-liposome had higher antitumor efficacy and lower side effects in comparison with other groups in the in vivo antitumor efficacy evaluation. No hemolysis phenomenon and histopathological changes were observed for SF/Gd-liposome. This preliminarily indicated that SF/Gd-liposomes had good biocompatibility and low toxicity under our experimental conditions.3. Preliminary Studies on SF/CE co-loaded liposomes (SF/CE-liposomes)The SF/CE-liposomes were prepared by the thin film hydration method. The optimal dose schedule of SF and CE was selected using MTT assay. Malvern Zetasizer Nano ZS instrument was used to determine the mean diameter, particle size distribution and zeta potential of the liposomes. The morphology of the liposomes was characterized by Transmission electronic microscopy. HPLC method was used to determine the encapsulation efficiency and drug loading of the co-loaded liposomes. In vitro drug release was assessed by the dynamic dialysis method. The size changes in different period of times were used to study the preliminary stability of SF/CE-liposomes. The synergistic antitumor effect of SF/CE-liposomes in cell level was evaluated using MTT assay. The in vivo antitumor efficacy was also evaluated on H22 tumor bearing mice to investigate the synergistic antitumor effect of SF/CE-liposomes.The lowest value of IC50 and CI were occurred at the same time when the molar ratio of SF/CE was 2:1. The simultaneous administration of SF and CE with a molar ratio of 2:1 was determined as the optimal dose schedule for later studies. The morphology of SF/CE-liposomes was spherical or ellipsoidal shapes and the mean particle size and PDI, zeta potential, encapsulation efficiency and drug loading were (173.8±4.0) nm and 0.123, (-14.4±1.0) mV, (89.6±2.0)%and (3.98±0.1)%, respectively. No apparent changes of the liposome sizes were observed in 30 d at 4℃ which indicated that the liposomes could remain intact in this condition. Good stability of SF/CE-liposomes was displayed in the tested medium including pH 7.4 PBS, cell culture media and normal saline with ten percent of plasma in it at 37℃. The SF/CE-liposomes showed slow and sustained release kinetics of SF and only 50% of the SF was released at approximately 60 h. The existence of CE in the SF/CE-liposomes didn’t affect the release kinetics of SF. In the in vitro cytotoxicity test, SF/CE-liposome (IC50=11.5±0.44) showed the highest cell cytotoxicity in comparison with the SF solution (IC50=13.09±1.49) and SF liposomes (IC50=22.67±3.90) in all the tested drug concentrations expect for the minimum concentration (p< 0.01). Significant difference (p<0.01) was also seen in the value of IC50. All these results demonstrated an obvious synergistic antitumor effect in vitro. The in vivo antitumor effiency of SF/CE-liposome was significantly higher than that of SF-liposomes and CE-liposomes (p<0.05), demonstrating the synergistic therapeutic effect in vivo.In conclusion, SF/Gd-liposomes and SF/CE-liposomes were constructed according to the principle of combination therapy. The properties such as physico-chemical properties, drug release, the anti-tumor effacy in vivo and vitro and other related properties in pharmaceutics of the two co-loaded liposomes were studied. The SF/Gd-liposome was engaged simultaneously in therapy and the precise monitoring of drug distribution and early feedback about the treatment’s efficacy with relatively low toxicity. The SF/CE-liposome could achieve better therapeutic effect by the synergy effect of SF and CE. The two co-loaded liposomes could significantly enhance the therapeutic efficacy, decrease the toxicity and show specific tumor accumulation ability compared with other experimental groups in this work, respectively. The two co-loaded liposomes showed great potential for HCC treatment. |
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