Study On Chitosan Microspheres Containing Docetaxel For Lung-targeting

Lung cancer is one of the most harmful malignant tumor to human health.Recently, the role of chemotherapy in lung cancer is no longer limited to the patients with inoperable advanced lung cancer, but also include systemic treatment of lung cancer as a comprehensive treatment program.However,the doses of chemotherapy drugs are large ,and they are lack of selectivity to tumor cell.they are killing tumor cells while also killing normal human cells, cause serious side effects, and brought great suffering to patients with cancer. The targeting drug delivery system of anticancer drug has been paid more and more attention with the deepening of tumor pathogenesis, targeting drug of new technologies, new carrier materials research.One of reaserches which the traditional first-line cytotoxic drugs that show exact effect are changed their pharmaceutical, pharmacokinetic properties, improved their orientation and selectivity to tissues,organs and tumors cells has become an important research direction of anti-tumor targeting drug delivery system. Docetaxel (DTX) is a semi-synthetic taxane anticancer drugs, which the mechanism is to strengthen the tubulin polymerization , inhibit depolymerization of microtubules, form stable non-functional microtubules, and destroy the mitosis and cell proliferation. Studies show that DTX have a exact role in inhibiting the growth of lung cancer cells.However, the current clinical injection distributes widely after administration, and non-targeting. It also solubilized by Tween 80 which causes severe adverse reactions.This study bases on the theory that 7 12μm microspheres can be filtrated mechanically by lung (pulmonary capillary diameter 3¹1μm) and concentrates in lung. In this study, DTX is prepared to microspheres which the diameter is in 7 12μm by optimizing the formulation and process .It mainly used to treat lung cancer because of the lung- targeting character. The preparation improves the efficacy while reduces the side effects of other tissues and organs.Objectives1. Investigate the formulation and process factors that influence the preparation of chitosan microspheres containing docetaxel (DTX-CS) for lung-targeting, and optimize the formulation and process.Prepare DTX-CS microspheres for lung- targeting with suitable size.2. Study comparatively between DTX-CS and docetaxel injection used in clinic in vivo distribution and lung targeting, provide the theory basis for the further development of DTX-CS microspheres preparations for lung-targeting.Methods1. Take chitosan as a carrier material, use emulsification-chemical crosslinking methord to formulate DTX-CS microspheres.Investgate the single factor (stirring speed, amount of crosslinking agent, chitosan concentration, solvent of docetaxel, etc.) according to the results of preliminary experiments.Optimize preparation process and formulation by orthogonal experiment, select the optimal process parameters, finally prove the repeatability of the process.2. Observe morphology, adhesions of microspheres by optical microscopy and take pictures of microspheres, measure the particle size , calculate average particle size and polydispersity index; Determine drug loading of microspheres by high performance liquid chromatography (HPLC), calculate the drug yield.3. According to China Pharmacopoeia(2010 edition), determine the cumulative release amount in vitro by dynamic dialysis method. First,get the media contain DTX at different time points;then determine the sample of DTX by HPLC after filtering them by 0.45μm microporous membrane.calculate the percentage of the average cumulative release amount of each time point, draw cumulative release rate-time curve.4. Kunming mouse were divided into DTX-CS microspheres group and docetaxel injection group, injected with DTX-CS and docetaxel injection by tail vein injection respectively. Obtain blood from eyeball of mice 5,15,30 minutes and 1,2,5,12,24,48,72 hours after administration.Then mice were killed by cervical disarticulation ,stripped and weighed heart, liver, spleen, lung and kidney quickly. Determine the DTX concentration in different organs by HPLC method,drew DTX concentration-time bar graph in each organs between two groups, compare DTX concentrations in lung between the two groups to find out whether DTX-CS microspheres are lung-targeting or not.5. The blood sample were filtrated by 0.45μm microporous membrane after disposing,and determined the concentration of DTX by HPLC, drew drug concentration-time curve in blood,compared the concentration of the two groups to find out whether DTX-CS microspheres are slow-releasing potential.6. Statistical Analysis All data were analyzed by SPSS 16.0 statistical analysis software. Measurement data are expressed as mean±standard deviation ( x±s). Differences among DTX injection group and DTX-CS group were analyzed by One-way ANOVA. LSD (Least Significant Difference) method should be used in the case of homogeneity of variance; otherwise Dunnett's T3 methods should be used in the case of variance nonhomogeneity. The sample data of the same organ at different time points were analyzed by two independent sample t-test analysis. P<0.05 was considered statistically significant. The date of DTX concentration in blood were analyzed by DAS 2.1.1 software.The main pharmacokinetic parameters of the two groups were also analyzed by two independent sample t-test analysis.Results1. We found the more significant factors which impact on the microsphere particle size, morphology, dispersion ,the drug loading are the chitosan concentration, amount of emulsifier, oil and water volume ratio, drug load ratio, amount of crosslinking agent, emulsification time and stirring speed from nvestgating the single factor.2. The DTX-CS microspheres obtained by the optimization of formulation and process are about round shape, smooth surface, no adhesion observed by microscope.The average particle size is (8.63±0.27)μm,the amount of microsph- eres diameter between 7and 12μm accounted for 83.5% of the total number of microspheres.The drug loading is (25.01±1.80) % and the drug yield is (85.54±2.21)%。3. The cumulative release rate-time curve shows that the microspheres obtained by the optimization of formulation and process release cumulatively(30.25±4.81)% at the beginning of 30minutes, which is consistent with Chinese pharmacop- oeia requirements. Then it shows a state of slow release, which the percentage of 24-hour cumulative release is(80.58±2.04)%。4. We can conclude that there were no diference of DTX concentration statistically significant among lung and plasma, heart, spleen, kidney by using one-way analysis of variance (one-way ANOVA) to analyze the injection group data. And DTX concentration in the plasma(10.637±15.803μg·mL^-1), liver (19.971±18.542μg·mL^-1), kidney (9.136±9.831μg·mL^-1)are also higher than lung (7.568±7.935μg·mL^-1). But DTX concentration in lung is significantly higher than plasma, heart, liver, spleen and kidney from microspheres group . And P < 0.05, The difference was statistically significant. Besides the DTX concentrations in lung of the microspheres group(29.184±18.736μg·mL^-1)is significantly higher than injection group(7.568±7.935μg·mL^-1),while DTX concentration in plasma, heart, liver, spleen and kidney are significantly less than injection group. The results shows that the microspheres group has certain lung-targeting property.5. Compared DTX concentrations in plasma of the two groups at different time po- ints after administration, DTX concentrations of the microspheres group were lower than the injection group at all the time points except 5,12h. The difference was statistically significan(tP<0.05). The differences of DTX concentration in plasma became smaller between the two groups with the time growth. the DTX concentration in plasma of microsphere group was not detected at 24h point after administration, as the concentration below the detection limit. However, the DTX concentration in plasma of injection group was 0.065±0.129μg·mL^-1. It showed that the concentration in plasma of the microsphere group was lower than that the injection group. The data of DTX concentration in plasma were analyzed by DAS 2.1.1 software. t1/2 of the two groups was 0.809h,5.633h respectively. The difference was statistically significan(tP=0.026). The average resident time of microspheres MRT were 1.949h and 2.526h, the difference was also statistically significant(P=0.049). It indicated that DTX microspheres showed a certain slow release.Conclusions1. The size of the microspheres made by the best formulation and process can satisfy the requirements of lung targeting; the preparation may become a new targeting therapy agent for clinical treatment of lung cancer.2. From the contrast experiment of DTX-CS microspheres and DTX injection, it can be preliminary considered that the DTX-CS microspheres obtained by this study maintain obviously lung-targeting, and the DTX-CS microspheres has a certain slow release in the lung of mouse.