Lung Cancer Combination Therapy:co-delivery Of Paclitaxel And Doxorubicin By Nanostructured Lipid Carriers For Synergistic Effect

Paclitaxel and doxorubicin are two representative medications that widely used in chemotherapy.Because of the unique drug mechanism, they were wide used in many combination chemotherapy of tumor. However, the free drugs used in clinical cancer therapy have lots of limitations such as high toxicity, low target capacity and easy to induce multidrug resistance. In the same time, both of them is indissolvable in water, so cause the configuration of drug is elaborate, and infusion of drug is complex. The produce technique of nanostructured lipid carrier is mature, nature of the drug is stable. The composition and structure of Nanostructured lipid carrier (NLC) make it have a wide range of drug inclusion, which means NLC can control the relief from small molecule chemical agents to the biological macromolecules. After encapsulation by liposome, the dynamic of drug changes a lot:prolonged half-life in the circulation, reduced side effects, and increased drug solubility. In present study, novel nanostructured lipid carriers for combination delivery of PTX and DOX were prepared by the melt-emulsification technique. In vitro cytotoxicity against NCL-H460 human non-small cell lung carcinoma (NSCLS) cell line was investigated, and in vivo anti-tumor of NLC was evaluated on mice models grafting NCL-H460 cells.Part Ⅰ Construction of PTX-and DOX-loaded NLCObjective:The composition and structure of Nanostructured lipid carrier (NLC) make it have a wide range of drug inclusion, which means NLC can control the relief from small molecule chemical agents to the biological macromolecules. After encapsulation by liposome, the dynamic of drug changes a lot:prolonged half-life in the circulation, reduced side effects, and increased drug solubility. NLC has the same structure with biological membrane, which means it can enter the blood circulation through many ways, increase the target capacity of the drug, and reduce the dosage of treatment. The aim of this reaearch is to develop a system that could achieve co-delivery of PTX and DOX, exert the ability of the two drugs, reduce the systemic toxicity, and get the significant better tumor inhibition efficiency.Methods:PTX-and DOX-loaded NLC (PTX-DOX-NLC) was prepared by melted ultrasonic dispersion method. The particle size (diameter, nm), polydispersity index (PDI) and surface charge (zeta potential, mV) were determined using a ZetaSizer Nano series Nano-ZS. The amount of DOX in microspheres was analyzed by a UV-Vis spectrophotometer at 480 nm. The amount of PTX in microspheres was analyzed utilizing a high performance liquid chromatography. The release behavior of DOX and PTX from NLC were assessed by the dialysis method.Results:The diameters from blank NLC to single drug-loaded NLC and PTX-DOX-NLC were around 125-130 nm. The PDI of PTX-DOX-NLC is 0.18. These results could be the evidence that the PTX-DOX-NLC has good dispersibility. The +27mV of potential could ensure the stability of the PTX-DOX-NLC and also facilitate the delivery of NLC to the negatively charged cancer cells. The drug loading content (DLC) and the drug loading efficiency (DLE) of PTX-DOX-NLC were only slightly decreased compare to PTX-NLC and DOX-NLC. These results prove that the PTX-DOX-NLC is very stable. The PTX and DOX in the PTX-DOX-NLC were released gradually over the 48-hour period following the Higuchi linear equation. 70% of PTX and DOX could be released in 24h. The in vitro release profile shows that PTX-DOX-NLC formulation has the capacity to release PTX or DOX at a sustained rate.Conclusions:1) In this study, PTX-DOX-NLC was prepared by melted ultrasonic dispersion method. The diameters from blank NLC to single drug-loaded NLC and PTX-DOX-NLC were around 125-130 nm. These results indicate that the adding of drug(s) did not affect the size of NLC.2) The DLC and DLE of PTX-DOX-NLC were only slightly decreased compare to PTX-NLC and DOX-NLC. However the stability of the PTX-DOX-NLC didn’t change. The PTX and DOX in the PTX-DOX-NLC were released gradually over the 48-hour period following the Higuchi linear equation. The PTX-DOX-NLC has the capacity to release PTX or DOX at a sustained rate.Part IIIn vitro cytotoxicity studiesObjective:The inhibition effect of drugs on human cancer cells is cytotoxicity test. The cytotoxicity is caused by the cell or chemical substances, but does not depend on the mechanism of apoptosis or necrosis. Cytotoxicity detection is mainly based on the change of cell membrane permeability and the most commonly used method is MTT. In this part, in vitro cytotoxicity against NCL-H460 human non-small cell lung carcinoma (NSCLS) cell line was investigated.Methods:MTT is a yellow dye. The MTT assay is a colorimetric assay for assessing cell metabolic activity. The detection principle is succinate dehydrogenase of mitochondria in living cells can make exogenous MTT deoxidize to blue insoluble purple crystal formazan and deposit in cells, whereas the dead cells can’t. Dimethyl sulfoxide (DMSO) could dissolve the formazan in cells. So the light absorption value at the wavelength of 490nm could be detected by enzyme linked immunosorbent assay instrument and the results could indirectly reflect the number of living cells. In a certain range, there is a positive correlation between the amount of MTT crystal formation and the number of living cells. This method is widely applied to the cytotoxicity test. Meanwhile it is characterized by high sensitivity and economy. This research MTT assay was used to investigate the cytotoxicity of drug loaded NLC and solution formulations in vitro. The drug concentration causing 50% inhibition (IC50) was calculated using Statistical Package for the Social Sciences version 17 software. To determine the optimal ratio of PTX and DOX in PTX-DOX-NLC, DOX and PTX combinations at various weight ratios were prepared. All samples were performed at different concentrations.Result:The viability of NCL-H460 cells treated with various concentrations of free drugs and drug(s) loaded NLC for 48 h decreased in a dose-dependent manner. The IC50 of PTX-DOX-NLC at the PTX/BCL weight ratio of 1/1 was the lowest compared with other weight ratios. Thus this ratio was determined. The cytotoxic effect of PTX-DOX-NLC on cancer cells was significantly higher than single drug NLC (3-fold), and free drug formula (9-fold) (P<0.05).Conclusions:MTT assay was used to evaluate the viability of NCL-H460 cells treated with various concentrations of free drugs and drug(s) loaded NLC for 48 h. Results showed the viability of tumor cells decreased in a dose-dependent manner. The IC50 of PTX-DOX-NLC at the PTX/DOX weight ratio of 1/1 was the lowest compared with other weight ratios. Thus this ratio was determined. The cytotoxic effect of PTX-DOX-NLC on cancer cells was significantly higher than single drug NLC (3-fold), and free drug formula (9-fold) (P<0.05). These results proved that PTX-DOX-NLC has the most robust and targeted anticancer efficiency.Part IIIIn vivo anti-tumor efficiency of PTX-DOX-NLC on nude mice models grafting NCL-H460 cellsObjective:SPF grade BALB/c (5-6 week old, body weight 18-22g) nude mice were widely used in basic medicine research like oncology, immunology, toxicology and clinical research. In vivo anti-tumor of PTX-DOX-NLC was evaluated on mice models grafting NCL-H460 cells.Methods:NCL-H460 tumor bearing nude mice model was established by subcutaneous injection. The tumor volume and appetite change were observed dynamically. Mice with NCL-H460 tumors were treated with PTX-DOX-NLC, PTX-NLC, DOX-NLC, free PTX, free DOX and tumor volume and body weight were measured every 4 days to evaluate the antitumor activities and systematic toxicities of various formulations. Tumor growth curve were drawing continuous for 21 days. HE staining was used for tumor structure examination. Statistical analysis was performed using the standard Student t test and one-way analysis of variance by SPSS 17.0 software. Data were presented as means of determinants (SEM) and P values< 0.05 were considered as statistically significant.Results:Tumor growth was significantly inhibited by PTX-DOX-NLC, than PTX-NLC and DOX-NLCs formulations (P<0.05). The obviously better tumor inhibition effects were observed in the NLC groups than the free drug solution groups. PTX-DOX-NLC exhibited the highest inhibition rate of 84%, followed by DOX-NLC (65%) and PTX-NLC (64%). Free DOX and PTX got about 26% tumor inhibition rates. Also, no obvious body weight change were found in three NLC groups; however, a progressive and irregular decrease in mean body weight was found in the free drug and saline group, with the reductively intake of foods, inactive in moving.Conclusions:Tumor growth was significantly inhibited by PTX-DOX-NLC, than PTX-NLC and DOX-NLCs formulations (P<0.05). The obviously better tumor inhibition effects were observed in the NLC groups than the free drug solution groups. PTX-DOX-NLC exhibited the highest inhibition rate of 84%, followed by DOX-NLC (65%) and PTX NLC (64%). Free DOX and PTX got about 26% tumor inhibition rates. The results suggested the best anti-tumor effect of double drugs contained NLC due to the synergetic effect of the two drugs, and the least systemic toxic side effect of the NLC formulations for the lung cancer treatment.