The Construction Of Nano-drug Delivery Systems Based On Amphiphilic Racemic Polypeptide Hybrid Copolymer

Amphiphilic block copolymers containing synthetic polypeptides have attracted much attention recently because of their excellent biocompability and technical ease.Generally speaking, existing studies mainly focus on levo polypeptides-based copolymers and their use in drug carriers’ construction. However, few investigations have focused on the self-assembly behavior and the potential application as drug carriers of racemic segments based hybrid polypeptides, such as PEG-poly (racemic amino acid) copolymers.Also, very few investigations have compared the self-assembly behavior and drug-loading process of racemic polypeptides-based polymers with their levo forms. Systematic and comprehensive investigations of racemic polypeptide segments in amphiphilic polymers are still needed to identify their role in nanostructure formation as well as to explore the potential applications of this nanostructure as a useful drug-delivery vehicle.In this study, mono or dual terminal aminolyzed polyglycol hydrochlorides with different molecular weight (mPEG5000-NH2·HCl、mPEG2000-NH2·HCl、NH2-PEG5000-NH2·2HCl) were synthesized using the classical Gabriel method.N-carboxy-leucine anhydrides with different chiroptical property (D-Leu-NCA,L-Leu-NCA and rac-Leu-NCA) were synthesized using triphosgene. The ultraviolet absorption test suggested that these anhydrides were unstable and could only stored in-20℃ with argon condition not longer than one week. In pretesting, the one-step method of polypeptides sythesis was established by directly using the rac-Leu-NCA as polymerization monomer, when comparing with the the existing two-step method which involves synthesizing the D-Leu-NCA and L-Leu-NCA monomer in separate steps and mixing them equivalently as racemic forms. Using this method, various polypeptides having different blocks’ composition and/or different amount of leucine residues, as the representative of racemic PEG-polypeptide copolymers, were synthesized via the ring-opening polymerization (ROP) of rac-Leu-NCA initiated by terminal aminolyzed polyglycol hydrochloride in anhydrous DCM with different feed ratios and characterized by IR, 1H-NMR, GPC and optical activity analysis.The self-assemble ability of these amphiphilic copolymers in aqueous was evaluated by traditional "pyrene fluorescence probe" method,and the obtained critical aggregation concentration (CAC) were between 0.9 and 7.2 mg/L depending on the different copositions. The extremely low CAC values implied the strong self-assemble tendency of these copolymers in aqueous conditions. Their self-assemble morphologies were studied using the dialysis. TEM photographs suggests that diblock mPEG (molecular weight: 2000 or 5000)-poly (racemic leucine) (PRL) have more tendency to form spherical micelles when polypeptides not too long, symmetrical triblock polypeptides (PRL-PEG5000-PRL)have more tendency to form polymersomes and asymmetry triblock polypeptides(PEG5000-PRL-PEG2000) could self-aggregate into polymersomes when peptides block relatively longer.Then, the in vitro cytotoxicity of the mPEG5000-PRL copolymer was studied. The cell viability in the presence of various concentrations of mPEG5000-PRL copolymer was in the range of 99.3±1.7% to 102.5±4.1% for MCF-7 cells and 99.0±1.8% to 103.7±2.7%for HUVEC cells. This result proved that mPEG5000-PRL copolymers exhibited almost no cytotoxicity against these two kinds of cells up to the concentration of 100 μg/mL,demonstrating the excellent biocompatibility. The novel docetaxel-loaded polymeric micelles based on mPEG5000-PRL copolymers were fabricated successfully using dialysis method by the micellization of the amphiphilic polymer in aqueous, with the highest drug loading content of 11 %. The hydrogen bonding effect, which was concluded to come from the interactions between docetaxel and free exposed amide groups due to the disordered racemic polypeptides, together with the hydrophobic interaction were believed to be the driving force to induce the formation of the drug-loaded micelles. This was also believed as the leading cause to the higher loading content of racemic polypeptides than corresponding levo polypeptides. And DTX-loaded mPEG5000-PRL micelles showed more toxic than the free drug against MCF-7 cells in a dose and time-dependent fashion.The even higher DTX-loading micelles based on mPEG5000-PRL copolymer were prepared using the film-hydration plus ultrasonic dispersion method with the drug loading content not less than 30%. And the optimum preparation method was established after the investigation and filtration of various conditions including film-hydration time/temperature, ultrasonic power/time, separation condition, feed ritos, and length of hydrophobic block etc. The high DTX-loading micelles (HD-micelles) prepared by this optimum method have the average size of 200 nm and Zeta potential of 1.2 mv. When added equivalent amount of PEG6000 (2.5%, w/v) and sucrose (2.5%, w/v), HD-micelles had excellent stability during lyophilization with 40 nm increasing in average size after reconstruction with water. HD-micelles also showed a sustained release property in pH 7.4 PBS, which released 77% of the loaded drug during 72 h and seldom suffered from the dilution by plasma. But in pH 5.5 PBS, the release rates of HD-micelles accelerated obviously. And HD-micelles also showed more toxic than the free drug against MCF-7 cells in a dose and time-dependent fashion. In addition, the operation characteristic of this method was believed to result to this exceptional high drug-loading ability by provide a more compact style to conjugate the polymer and drug.Finally, the UPLC-MS/MS method was established to determine DTX concentrations in plasma and tissues. With this method, the pharmacokinetic and tissue distribution characteristics of HD-micelles after intravenous injection were evaluated in wistar rats and Kunming mice, respectively. The in vivo results showed that, t1/2 of HD-micelles was significantly longer than DTX-solution at the dose of 5 mg/kg (P<0.05), AUC0-t, CL and Vss were all significantly different to DTX-solution at the dose of 5 and 10 mg/kg. The lung targeting effect was also observed in HD-micelles during the tissue distribution test at the dose of 5 mg/kg. Ascite S180 rat cells were applied as tumor model to study the antitumor activities of HD-micelles, and the results showed that micelles had better antitumor effect than DTX-solution at the dose of 5 mg/kg, and similar powerful antitumor activity with DTX-solution at the dose of 10 mg/kg.