Studies On PH-sensitive Micelles Of Poly (2-ethyl-2-oxazoline)-poly (D, L-lactide)

On the basis of related copolymer synthesis and investigation of its property, Amphiphilic block copolymers Poly (2-ethyl-2-oxazoline)-Poly (D,L-lactide) (PEOz-PDLLA) with suitable EOz/DLLA molar ratio were synthesized by two-step ring-opening polymerization and characterized by FTIR,’H-NMR and gel permeation chromatography(GPC). The number-average molar mass (Mn) of the copolymers, determined by GPC, were 1.76x 104 and 2.87×104, with a Mn of 7.11×103、1.24×104 for each hydrophilic segment, respectively. And the ratio of the block segments is similar. The copolymer solubility test suggested that the copolymers had good solubility in most solvents but saturated hydrocarbon. The pKa of PEOz1、PEOz2、PEOz1-PDLLA1、PEOz2-PDLLA2 from potentiometric titration was 6.42、6.28、6.52、6.41, respectively, which suggested PEOz-PDLLA would show pH-sensitivity under weak acid conditions. The critical micellar concentrations (CMC) of the copolymers were measured by pyrene fluorescent probe technique. The results showed that the CMC of PEOz1-PDLLA1、PEOz2-PDLLA2 in solution of pH 5.0 to pH 7.4 were range in 1.2×10-3 g·L-1 to 5.1×10-4 g·L-1 and 1.0×10-3 g·L-1 to 3.1×10-4 g·L-1, respectively. The copolymer could self-assembly into core-shell structure micelle with hydrophilic segment outward and hydrophobic segment inward. The particle size of the micelle was about 26 nm, which increased with the pH value decreasing.Docetaxel was chosen as the model drug, and film dispersion method was used here to prepare the micelle. By preparing micelle in different conditions, we investigated the influence of formula factors on entrapment efficiency, established the optimal formulation as follow:10 mg of PEOz-PDLLA, 1mg of docetaxel, 1mL of dichlormethane, and 2 mL of pH 7.4 PBS. The entrapment efficiency of PEOz-PDLLA micelle reached 94.88%, and the drug-loaded amount was 8.67%. The mean diameter of micelles was (35.3±4.9) nm. The in vitro drug releasing behavior was studied by dialysis. The drug releasing of micelle was accelerated in the medium with pH decreasing. The cumulative drug releasing of micelle in the medium with pH of 7.4 and 5.0 at 72h was 57.16% and 83.85%, respectively. When the drug-loaded amount reached 16.19%, the micelle stability decreased and there was an obvious burst release. The release test showed the release of drug from the micelle was non-Fick diffusion mechanism, and in weak acid condition diffusion-controlled mechanism was more obvious. Hemolysis test, administration position stimulative assay and eye irritation assay of the copolymer were studied with physiological saline as control. The protective effects of sucrose、glucose、mannitol、trehalose were investigated. Finally the combination of mannitol and trehalose both at a concentration of 3% was chosen. We established an assay method for docetaxel in plasma, and studied the pharmacokinetic properties of PEOz-PDLLA micelle. Compared with docetaxel solution, docetaxel PEOz-PDLLA micelle administrated by i.v. could significantly extend the circulating time of doctaxel in plasma, and reach sustained-release effect. The AUC、MRT and t1/2 of doctaxel PEOz-PDLLA micelle was respectively 4.8,32.4 and 28.7 times of that of the docetaxel solution group. While the CL of docetaxel solution group reached 4.3 times of that of the docetaxel micelle group, which showed the long-term circulating capability of PEOz-PDLLA micelle.Finally, we established an assay method for docetaxel concentration in tissues. The distribution of docetaxel in tissues was determined after i.v. injection of docetaxel solution and micelle solution into the tail vein of rats. Results showed that docetaxel solution distributed rapidly and extensively, could somehow penetrate the blood brain barrier, and cause acute and chronic cardiac toxicity. The distribution of DTX micelle was accomplished in 2 hours, with DTX targeting ability enhancing in liver and spleen. Meanwhile distribution in hear、brain and kidney were decreased, which could depress DTX side effects on these organs.