| Paclitaxel, extracted from Taxus brevifolia, is a new type anticancer drug, which has been widely used for the treatment of various cancers including ovarian, breast, head and neck, colon, prostate and non-small cell lung cancer. Due to its poor solubility(about0.25μg/mL), paclitaxel is stabilized and dissolved in a1:1(v/v) mixture of dehydrated alcohol and Cremophor EL(polyethoxylated castor oil). However, Cremophor EL produces severe hypersensitivity reactions, neurotoxicity, nephrotoxicity, labored breathing, lethargy and hypotension, which hinders its clinical application. Investigating oral delivery system of paclitaxel could not only reduce side effects, but also greatly improve compliance of patients. But, the oral bioavailability of paclitaxel is very low. Apart from low aqueous solubility, several reports indicated that P-glycoprotein(P-gp) hinders the transport of paclitaxel from the gut.Polymeric micelle is a new kind of drug delivery system, which could increase solubility and bioavailability of insoluble drugs. Pluronic block copolymers are very promising drug carrier for preparation of polymeric micelles given their non-toxic, non-stimulant, non-immunogenic and good solubility in body fluids. Moreover, it has demonstrated that Pluronic block copolymers could change the structure of the cell membrane and suppress the activity of P-gp to enhance the transmembrane diffusion of P-gp substrates. Hence, Pluronic F127(PF127,PEO106-PPO70-PEO106) and P123(PP123,PE02o-PP065-PE02o) were selected as the carriers to prepare polymeric micelles for oral delivery system of paclitaxel. In an attempt to further improve the absorption of paclitaxel by prolonging the contact time between the preparation and gastrointestinal tract, poly(acrylic acid)(PAA), a promising kind of bioadhesive materials, was conjugated to hydroxyl group of PF127.The major works were as follows:1. Synthesis of Pluronic F127-poly(acrylic acid)(PF127-PAA)A type of copolymer composed of PF127and PAA was synthesized using N-(3-Dimethylaminoprop-yl)-N’-ethylcarbodiimide hydrochloride(EDC) as catalyst. And polymeric micelles formed with PF127-PAA polymer are pH-sensitive. In order to obtain the optimal condition, with the critetions of the optical transmittance in varying pH values and the drug release behavior in vitro(pH=6.8), paclitaxel-loaded PF127-PAA micelles were prepared to evaluate the effect of the mass ratio of PF127and PAA on synthesis of PF127-PAA. The results indicated that when the mass ratio of PF127and PAA was fixed to1:1, the optical transmittance was changed significantly and the drug released most rapidly. So the mass ratio of PF127and PAA was fixed to1:1.The PF127-PAA polymer was characterized by FT-IR and1H-NMR. In the FT-IR spectrum of PF127-PAA, the two characteristic peaks at1723.89cm-1and1109.84cm-1attributed to-C=O stretching mode and C-O-C stretching mode respectively were observed while the only characteristic peak at1112.10cm-1corresponding to-C=O stretching mode in PF127spectrum and the only characteristic peak at1709.76cm-1corresponding to C-O-C stretching mode in PAA spectrum were observed, which could confirme the synthesis of PF127-PAA polymer.In the1H-NMR spectrum, the peaks used to determine the PF127-PAA polymer were the one attributed to PEO methylene protons of PF127at3.36ppm, the peak attributed to the protons of PPO methyl pendant groups of PF127at1.13ppm and the peak that are characteristic of methylene protons of PAA at2.19ppm.The content of PAA in PF127-PAA copolymer was measured by acid-base titration(22%); The critical micelle concentration(CMC) was determined by fluorescence probe technique using pyrene as a hydrophobic probe(2.9mg/mL).2Preparation and characterization of paclitaxel-loaded PF127-PAA/PP123polymeric micellesThe thin-film hydration method was used to prepare paclitaxel-loaded PF127-PAA/PP123polymeric micelles. The amount of PF127-PAA was fixed to50mg. Based on the results of single factor experiments, feeding of paclitaxel(X1, mg), amount of PP123(X2, mg), hydration temperature (X3,℃), volume of water phase (X4, mL), pH of water phase (X5) were optimized employing the uniform design with five factors and three levels. Taking the drug loading as evaluation index, the final optimized formulation was2mg of paclitaxel,30mg of PP123,55℃of hydration temperature,4.1mL of water phase,4of water pH. The drug loading was (1.88±0.03)%; the micelles exhibited spherical shape and ranged in uniform size; The particles size was (23.7±1.3)nm; The zeta potential was (1.54±0.05)mV.The dialysis method was used to investigate in vitro release profiles of paclitaxel from polymeric micelles. The results showed that the drug release behavior from free paclitaxel fit first order kinetics model and95.68%of paclitaxel had been released in ten hours. The drug release behavior of PF127-PAA/PP123micelles and PF127/PP123micelles fit Weibull model and the correlation coefficient was0.9964,0.9969, respectively. In comparison with paclitaxel-loaded PF127/PP123micelles, the paclitaxel-loaded PF127-PAA/PP123micelles released paclitaxel faster, indicating that PAA could facilitate release of paclitaxel from polymeric micelles.3In situ absorption property of paclitaxel-loaded micelles in rat intestineAn in situ intestinal perfusion technique in rats was used to study the absorption property of paclitaxel-loaded micelles.The absorption rate constant (Ka) of paclitaxel-loaded PF127-PAA/PP123micelles with low, middle, high dose were0.0790h-1,0.0803h-1,0.0787h-1, respectively, and the correlation coefficient were0.9402,0.9857,0.9755, respectively. The results indicated that the mechanism of paclitaxel-loaded micelles in intestinal absorption was passive diffusion, showing first order kinetics.The Ka of paclitaxel solution, paclitaxel-loaded PF127-PAA/PP123micelles and paclitaxel-loaded PF127/PP123micelles were0.0107h-1,0.0787h-1,0.0691h-1, respectively. The absorption speed of the two kinds of micelles was faster than that of paclitaxel solution which suggested that micelles could improve absorption of paclitaxel. This paper also investigated the impact of P-gp inhibitor verapamil on intestinal absorption of paclitax el and the results indicated that pluronic micelles could suppress the activity of P-glycoprotein to enhance the absorption of paclitaxel.The test of main segments indicated that paclitaxel-loaded PF127-PAA/PP123micelles could be absorbed by all segments of the intestine. The absorption percentages of duodenum, jejunum, ileum and colon were (24.06±2.91)%,(8.77±2.54)%,(3.73±1.07)%,(16.41±3.55)%, respectively. The main segments of paclitaxel-loaded PF127-PAA/PP123micelles absorbed in intestine were duodenum and colon.4. Pharmacokinetics studies in ratsBased on the results of in situ intestinal perfusion experiment, pharmacokinetic experiment of paclitaxel-loaded PF127-PAA/PP123micelles was carried in rats. In contrast to paclitaxel injection, the peak concentration(Cmax) and the area under the concentration-time curve(AUC) were improved from80.70μg/L,681μg/L*h to90.64μg/L,1370μg/L*h, respectively; the biological half-life(t1/2) and the mean residence time(MRT) were prolonged from5.46h,8.46h to8.93h,13.66h, respectively. These results showed that PF127-PAA/PP123micelles could increase absorption of paclitaxel by oral administration. |
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