Overcoming Multidrug Resistance By Doxorubicin Nanocarriers

Drug resistance is a major problem that limits the effectiveness of chemotherapies used to treat cancer. There are a variety of strategies and approaches to inhibit or circurnvent MDR, including chemical agents which reverse increased efflux such as verapamil, new anticancer drugs that are not substrates of drug transporters, strategies on biotechnology such as gene treatment. But it is one of the significant missions in the area of overcoming MDR. Drug carrier is a novel approach to overcoming MDR in cancer nowadays. The main aims of this research were to investigate nanocarriers to overcome MDR in cancer, based on doxorubicin, which will provide the basis for future studies of overcoming drug resistance and ultimately impoving chemotherapy and the outcome of cancer patients.The contents of this thesis were including (1) Preparation and characterization of liposome, SLN and niosomes of doxorubicin; (2)evaluation of overcoming MDR effect of the nanocarriers with the resistanct cnacer cells K562/DOX; (3) evaluation of overcoming MDR effect of the vehicles such as non-ionic surfactant, cationic with K562/DOX cells; (3)investigation of the mechanism of overcoming MDR with the resistanct cnacer cells K562/DOX; (4)study of Phamacokineties, biodistribution and pharmacodynamics of the liposome and niosomes of doxorubicin.The model of resistant cell and determination of doxorubicin in cells were established. The K562/DOX cells expressed high activity of P-glycoprotein(P-gp) and showed an obviously resistance to K562/DOX. The resistance index(RI) of K562/DOX cells was up to 107.90, and Verapamil(VER) as P-glycoprotein substrate could partially reverse this resistance. The results of uptake of DOX further proved that K562/DOX cells had significant resistance to DOX, and VER can partially reverse the resistance and increase the uptake of drug in resistant cells. The method of spectrofluorometer was set up to determine the concentration of DOX. The assay exhibited a linear range of 10-1000 ng/ml and gave a correlation coefficient (r) of 0.9998 or better. The intra-assay precision , inter-assay precision , recovery were validated according to the FDA guidelines on bioanalytical method validation. It is effective to avoid the fluorescence quence when incubated with 0.1% SDS during determination.Liposome can partially overcome multidrug resistance. Doxorubicin liposome (DOX-L) was encapsulated by the ammonium sulfate gradient-driven method. It is stable and the leakage is slow. DOX-L enhanced the drug accumulation in K562/DOX cells, and improved DOX retention in cells after withdrawal. It showed that liposome can improve the intracellular DOX concentration, which showed DOX-L can partially overcome the drug resistance. The uptake of DOX-L was energy-dependent and was influenced by temperature. Endocytosis inhibitors (sodium azide, mannitol and chloroquine) decreased significantly accumulation of DOX (p<0.01). The result revealed that DOX-L was uptaken through the endocytosis of energy-dependent.To study the intracellular kinetics behavior and distribution of DOX, reversed-phase HPLC mothod was established to determine nuclear DOX concentration. The cellular uptake kinetics study showed that DOX-L could significantly increase intracellular accumulation and nuclear delivery of DOX-L compared with DOX-I at same dose. The nuclei levels of liposome rose slowly and reached the plateau after 2h incubation, whereas the free drug reached the plateau in 15min, suggesting that it takes times for the liposome to transport from cytoplasm to nuclei. Our results also demonstrated that liposome extended the retention in nuclei when exposed to resistance cells.Pharmaceutical technique of modifying on the surface of liposomes will have effect on cellular uptaken and effluxed and overcoming MDR. A series of non-ionic surfactnat including Pluronic (F68, F87, F88, F108, P85), Span (20, 40, 60, 80, 85), Tween (20, 80) and Cremophor EL were incorporating into DOX-L. The effect of expicients on the properties of liposomes and cellular uptake were investigated. Results showed that non-ionic surfactant modifying DOX-L caused significant increasing of cellular uptake. And after 2 h exposure to non-ionic surfactant modified DOX-L, the intracellular DOX concentration was correspond to HLB of non-ionic surfactant. The intracellular DOX level of different series non-ionic surfactant modified DOX-L was not only correlate to the HLB, but to property of surfactant.The cationic liposomes increased DOX allocate into nuclei but decreased the drug amount in cells. The cationic liposomes were prepared by DC-Choi or CTAB. We found that, when compared to DOX-L, the drug amount in intact cells decreased, but the DOX concentration in nuclei was similar to that of DOX-L. Percentage of nuclei in k562/DOX cells exposed to the cationic liposomes was high than that of DOX-L. The IC₅₀ of DOX-L-DC-Chol and DOX-L-CTAB was 8.629±0.808μg and 9.653±0.889μg, repectively, lower than that of DOX-L(15.475±2.392μg).There was synergistic effect of DOX-L and verapamil on overcoming MDR. In order to estimate the influence of verapamil(VER) on the uptaken and effluxed of DOX-L, a new liposome(DOX-L-LV) co-encapsualting DOX and VER into liposome was developed. The data of DOX-I-FV demonstrated a rapid accumulation and reached a plateau within 0.75h, whereas in cells treated with DOX-L-FV, the cellular DOX levels continued to increase up to 2 h. After 2 h of treatment, nearly 1.25-fold of DOX delivered by DOX-I-FV was transported into cells compared that produced by DOX-L-LV. The amount of drug uptaken by K562/DOX cell exposed to DOX-L-LV was significant lower than that of DOX-L-FV at 2 h, but it continued to increase and get close that of DOX-L-FV at 4 h. The IC50 of DOX-L-LV as measured by MTT assay in K562/DOX cells was 0.510±0.012μg/mL, 5.4 fold less than that for liposomal DOX with free VER (DOX-I-FV 2.739±0.457μg/mL), but only about 1.6 times less than DOX-L-FV(0.821±0.182μg/mL). The IC₅₀ cytotoxicityon K562/DOX cells of the various formulations was as follows: DOX-L-LVmax of DOX-L-Pluronic P85 in tumor were 4.65 and 1.44 folds higher than that of DOX-I, respectively. The relative tumor tissue exposure (Re) (contain heart, kidney, liver, spleen, lung, interine) were above 1 and the heart, kidney tumor targeting efficient were above 10. It showed that DOX-L-Pluronic P85 could reduce the mice cardiac and renal toxicity compare with DOX-I. Pharmacological test showed that DOX-L-Pluronic P85 at 2 mg/kg induced a 30.83% tumor inhibition rate, Similar with DOX-I at same dose showed tumor inhibiton rate of 32.09%. DOX-L-Pluronic P85 at 1 mg/kg and 4 mg/kg had tumor inhibiton rate of 28.53 % and 51.91 %, respectively. The latter was about 2 folds higher than that of DOX-I at 2 mg/kg. Pathologic slice of tumor tissue also showed the effect of antitumor was higher than that of DOX-I, and the effect of antitumor was lower than that of DOX-I.Solid lipid nanoparticle (SLN) could effectively overcome MDR. We developed a SLN preparation which attempted the use of organic anions to form ion pairs with Dox for the improvement of drug lipophilicity and drug loading. SLN could significantly improve DOX accumulated in K562/DOX cells. And we were interested to find that SLN uptaken by K562/DOX relatively slowly and effluxed also slowly. It showed that SLN could improve DOX accumulated in resistant cells and SLN could partially overcome MDR. We prepared 3 different SLN by different non-ionic surfactant: Pluronic F87, F88 and P85, repectively. And found that the DOX concentration when K562/DOX cells exposed to DOX-SLN-P85 was more than that of DOX-SLN-F87 and DOX-SLN-F88 at different time point. It might because the EPO of Pluronic P85 was smaller that that of Pluronic F87 or F88, which might influence the fluidity of cellular membrane when SLN incubated with K562/DOX cells. The uptake of DOX-SLN was also through the endocytosis of energy-dependent and was influenced by temperature.DOX niosomes could also partially overcome MDR. DOX niosomes were prepared by film dispersion method. The amount of drug uptaken by K562/DOX cell exposed to DOX niosomes was significantly higher than that of Dox solution (p < 0.05) at the same drug concentration. After 4 h exposure to DOX-N, the intracellular DOX concentration was 1.4 times higher than that incubated with free drug. The uptake of DOX-N was also energy-dependent and was influenced by temperature. Endocytosis inhibitors (sodium azide, mannitol and chloroquine) decreased significantly accumulation of DOX (p<0.01). The result revealed that DOX-N was uptaken through the endocytosis of energy-dependent.