| Paclitaxel (Taxol), which is extracted from Taxus, is the anticancer agent of taxane structure. PTX has the function of stabilizing microtubule, inhibiting mitotic division and inducing apoptosis, and is an efficient cells poisonous drug. In December 1992 the FDA approved Taxol for refractory ovarian cancer. Later, it is approved for breast cancer. Today it is successfully used for a variety of cancers, including ovarian, breast, non-small-cell lung and Karposi's sarcoma. PTX is the golden chance in the treatment of cancers in the world.The study of physico-chemical property showed that solubility of PTX was only about 0.25μg/ml.It was easy to dissolve in n-octanol and had a good liposolubility. To enhance its solubility is one of the key factors in PTX extensive use. Presently, the only available commercial preparation of PTX is Taxol, a concentrated solution composed of a 50:50 (v/v) mixture of Cremophor EL (polyoxyl 35 castor oil) and dehydrated alcohol, which is used for breast and ovarian cancer. Unfortunately, serious side effects, such as hypersensitivity, nephrotoxicity, neurotoxicity, labored breathing, lethargy and hypotension, attributable to Cremophor EL have been reported, so its clinical application is extremely limited. Because formulations of the oil needle has not been in clinical application, the using technologies of liposomes and nanoparticles in current declaring water injection solve the problem of low water solubility.For these reasons, to enhance water-solubility, targeting and reduce side effects of Paclitaxel, we use Pluronic F127 consisting of polyoxyethylene-polyoxypropylene-polyoxyethylene triblock polymer as carries, which can form spontaneously micelles above the critical miceller concentration (CMC) in water. The structure of micelles is well described by a core-shell structure with the hydrophilic shell (PEO) and the hydrophobic core (PPO). Poorly water soluble Paclitaxel can be solubilized in hydrophobic core of micelle. Because the particles with positive charge are easy to target to lung tissue, the stable micelles were prepared by cross-linking with low molecular weight polyethylenimine (PEI) and Pluronic F127 to increase lung targeting and overcome the resolved phenomenon of micelles after dilution into the systemic circulation system. In order to further enhance active targenting and stability,the F127-FOL, which the folic acid (Folic acid, FOL) binds into the end of F127, was used to form mixed micelles with F127-PEI at a certain mass ratio (1:9).The subject of the research process and the results were as follows:1. F127-PEI polymeric micelles were prepared with primary amino end of low molecular weight PEI and CDI-activated Pluronic F127 to cross-link by the emulsification/solvent evaporation method. According to the preparation process of the emulsification/solvent evaporation method, the drug loading coefficient and entrapping ratio were taken as main criterions, and the critical micelle concentrtion (CMC) as the secondary criterion to evaluate the effects of activated-F127 and PEI mass ratio, rotary evaporation temperature, the water phase volume and emulsifying time to determine the optimal preparation. As a result, the optimal formulation and process by the single factor experiment as follows:activated-F127 and PEI mass ratio was 25:1, rotary evaporation temperature was 30℃, the water phase volume was 10ml, emulsifying time was 3 min.F127-FOL was synthesized by CDI-activated FOL and the hydroxyl end of F127.The characterization of these synthesized carrier material structure was investigated. In the FT-IR spectrum,1761.54 cm"'peak of the carbonyl imidazole may indicate the formation of activated-F127; the characteristic peaks at 3422.43cm-1 and 1717.16cm-1 were attributed to amino group and carbonyl group of F127-PEI copolymers respectively. In1 H-NMR spectra of F127-FOL copolymer,δ:1.03 (CH3in PPO of F127),1.1-1.48 (m, CH2CH2CO of folate),3.33 (H2O),3.42-3.48(s, CH in PPO of F127),3.50 (s, CH2CH2O of F127),3.6 (d,CH2in PPO of F127),4.5 (d, NCHCO of folate),6.6 (d,3,5-H of benzene ring in folate),7.6 (d,2,6-H of benzene ring in folate),8.6 (s, C7-H in pteridine proton of folate), which can be characterized F127-FOL copolymer structure.The content of PEI and folic acid in F127-PEI and F127-FOL were measured respectively by UV spectrophotometer to verify the experiment grafting PEI and folic acid.2. PTX-loaded polymeric micelles were prepared with F127-PEI copolymer by thin-film hydration method. Based on the results of single factor experiments,with the evaluation index of the drug loading coefficient and entrapping ratio, micelle formulation was optimized employing the central composite design-response surface methodology with four factors and five levels.The final optimized formulation was 2.5 mg of PTX, lOmL of water phase,6.9 of water pH,32.5℃of hydration temperature. PTX-loaded F127-PEI/F127-FOL mixed micelles were prepared according to the final optimized formulation. Study of physical and chemical properties of micelles showed that two kinds of micelles were shperically shaped, a slight cross-linking and ranged in uniformity size. For F127 micelles, F127-PEI micelles and F127-PEI/F127-FOL mixed micelles, their average particles size and Zeta potential were 19.19,56.45,68.77nm, and-9.49,7.67,-5.20 mV respectively.3. The drug release behavior from PTX-F127-PEI copolymer micelles and PTX-F127-PEI/F127-FOL in vitro could promote the release of PTX, which attributed to a certain pH response of the structure of PEI and folic acid in both micelles. As a result,PTX release of the cross-linked micelles with a pH response is faster than the non-crosslinked micelles in the same pH condition.4. The effects of PTX-F127 micelles, PTX-F127-PEI micelles and PTX-F127-PEI/ F127-FOL mixed micelles on the growth of HeLa carcinoma cell in vitro were investigated using MTT colorimetric method to evaluate the antineoplasmic activity in vitro. As a result, the antineoplasmic activity of PTX in vitro was PTX-F127-PEI/ F127-FOL mixed micelles>PTX-F127 micelles>PTX-F127-PEI micelles> PTX solution and IC50 of four preparation groups were 0.425,1.106,1.776 and 17.781μg/ml resepectively. PEI are cytotoxic due to their high buffering capacity that may cause endosomal break-up, so the cytotoxicity of empty F127-PEI polymeric micelles was investigated by MTT colorimetric method. The results showed that the inhibition rate of empty micelles decreased with the reduction in the amount of low molecular weight PEI. But the inhibition rate was less than 6%, far less than the high molecular weight PEI (25kDa) toxicity.5. The pharmacokinetic experiment of Taxol injection, PTX-F127-PEI micelles, PTX-F127-PEI/F127-FOL mixed micelles was carried out on mouse, and DAS 2.0 pharmacokinetic procedure was applied to calculate parameters and choose the best compartment model. Three preparations were all fitted two compartment model after i.v. administration, pharmacokinetic equations of Taxol injection, PTX-F127-PEI micelles, PTX-F127-PEI/F127-FOL mixed micelles were respectively C=1.449e-5.076t+0.438e-0.147t, C=60.960e-14-447t+0.736e-0.032t, C=1.449e-2.342t+ 0.273e-0.022t. Compared with the pharmacokinetic parameters of three preparations, the AUC and t1/2βof PTX-F127-PEI micelles and PTX-F127-PEI/F127-FOL mixed micelles (13.380 (mg/L)*h,5.868 (mg/L)*h) and t1/2β(21.444,31.135h) were significantly higher than Taxol injection (2.767 (mg/L)*h,4.963h. While drug clearance (CL) of micelles (0.095L/h/kg for PTX-F127-PEI micelles and 0.415L/h/kg for PTX-F127-PEI/F127-FOL mixed micelles) was less than Taxol injection (0.792L/h/kg); MRT of micelles (9.404h for PTX-F127-PEI micelles and 9.586h for PTX-F127-PEI/F127-FOL mixed micelles) was longer than Taxol injection (2.533h). The results indicated that PTX-F127-PEI micelles and PTX-F127-PEI/F127-FOL mixed micelles could significantly lengthen the retention time of drugs in vivo and had a well-sustained release efficacy.6. The tissue distribution of PTX-F127-PEI micelles, PTX-F127-PEI/F127-FOL mixed micelles and Taxol injection was investigated after i.v. administrations into mouse. Compared with Taxol injection, those micelles distinctly changed the distribution of PTX in vivo and increased the concentration of drug in target organs greatly. The overall targeting efficiency (Te) of micelles in lung was higher than Taxol injection, the relative taking efficiency (re) were 3.687 and 3.119 respectively,the maximum concentrations of lung were enhanced by 4.256 times and 3.720 times. Te of PTX-F127-PEI/F127-FOL mixed micelles with the significant lung targeting was higher than PTX-F127-PEI micelles, but re and the maximum concentrations (Ce) were less than PTX-F127-PEI micelles. The relative taking efficiency (re) of PTX-F127-PEI micelles and PTX-F127-PEI/F127-FOL mixed micelles in plasma were 8.879 and 2.742 respectively, while Ce of liver, spleen and kidney were all less to 1. In conclusion, micelles can change the distribution of PTX in tissues and organs and enhance the lung targeting. |
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