Studies On The TFu-loaded Liposomes

Chemotherapy has been one of the three major treatment methodologies for malignant tumors, while the significant side effect and the low stability of the therapeutic agents decrease the therapeutic efficiency, decline the compliance of the patients, and limit the utilization of the chemotherapy. Prodrugs are one of the strategies to improve the parmaceutical properties and therapeutic efficiency of the anti-cancer agents. A prodrug is a pharmacological substance which is administered in an inactive or significantly less active form. Once administered, the prodrug is metabolized in vivo into its active form, accordingly alter the therapeutic index by the improved efficacy and stability as well as the reduced toxicity.N₃-O-toluyl-Fluorouracil (TFu) was synthesized and evaluated as one of the newest effective prodrugs of 5-Fu in our group and its strong anticancer activity had been demonstrated in GC1901 and NKM-45 cell lines and mice. It was expected to exhibit tumor-target therapeutic effect by selectively releasing 5-Fu in tumor tissues. However, the clinical application of TFu might be limited due to its poor aqueous solubility and the low stability.In the present study, TFu was taken as the model drug to be wrapped up into liposomes by modern nano-drug delivery technique. TFu-loaded liposomes were characterized according to particle size and size distribution, zeta potential, drug entrapment efficiency, drug loading and the physical stability, respectively. In vitro release and in vivo pharmacokinetic characteristics, the bioavailabilities, the tissue distribution and the effects of the liposomes' sizes were investigated in mice, which provided experiment and theoretical basis for utilizing liposomes in malignant tumor chemotherapy. The enhancing absorption mechanism of the improving drug bioavailabilities by utilizing liposomes was discussed in order to develop more efficient and clinical practical oral liposomes formulation. The main methods and results were as follows:1. Determination of entrapment efficiency and drug contentProtamine aggregation method was applied to separate free TFu and the TFu-loaded liposomes. The drug concentration was then determined by HPLC method.2. Formulation optimization and preparation of TFu-loaded liposomesThe entrapment efficiency was taken as the index to evaluate the effects of the weight ratio of drug to lipids, weight ratio of soya lecithin to cholesterol, volume ratio of ether to phosphate buffer and bath temperature on the characteristics of liposomes. The weight ratio of drug to lipids, weight ratio of soya lecithin to cholesterol, and the volume ratio of ether to phosphate buffer, were all chosen as the most influential factors, which were optimized by uniform experimental design concerning theentrapment efficiency.3. The physicochemical characteristics, in vitro drug release and stability of TFu-loaded liposomes as well as the preparation of lyophilized TFu-loaded liposomesFour different sizes (530, 400, 320 and 180nm) of TFu-loaded liposomes were obtained by high-pressure homogenization for 5 times at pressure of 10bar, 80bar, 200bar and 600bar, respectively. The zeta potentials were -6.76, -6.64, -6.44 and -6.56 mV, and the polydispersities were 0.481, 0.448, 0.366 and 0.093, respectively. The drug entrapment efficiencies were (88.5±0.25)%, (85.6±0.41)%, (88.7±0.32)% and (80.0±0.45)%, and the drug loading were (8.9±0.2)%, (8.9±03)%, (8.6±0.4)% and (7.9±0.4)%, respectively.The in vitro release of TFu from the solution (in acetonitrile-water, 50:50, V/V) and from liposomes with 400nm size in phosphate buffered saline (pH7.4), artificial intestinal juice and artificial gastric juice were evaluate using the dialysis bag diffusion technique, respectively. TFu-loaded liposomes all presented controlled release properties, and nonlinear fits of TFu release from liposomes and solution were modeled. Both in phosphate buffered saline (pH7.4) and artificial intestinal juice, the in vitro release behavior of TFu from liposomes could be described by double phase kinetics model and expressed by the following equations: 10O-Q=98.18e^-0.2735t+40.89e^-0.028t(Ra=0.9990,R_β=0.9963) and 100-Q=96.00e^-0.2934t +26.53e^-0.027t, R_α=0.9941, R_β=0.9931, respectively; While in artificial gastric juice, the TFu release behavior from liposomes in vitro was in accord with Weibull model (ln[-ln(1-Q)]=0.804lnt-0.88, r=0.9915). In case of solution, all the release profiles of TFu could be described by first order kinetics model and could be expressed by the following equations: ln(100-Q)=-0.5585t+4.037 (r=0.9984),ln(100-Q)=-0.7151t+ 4.2232(r=0.9965) and ln(100-Q)=-0.567t+ 4.1207(r=0.9970), respectively.The stability test indicated that leakage rate of TFu from liposomes (400nm) reached 37.77% at 4°C after 12 months. To improve the stability of liposomes, lyophilized TFu-loaded liposomes were prepared by adding sufficient quantum of mannitol into the homogeneous liposomes suspension (ratio of mannitol to lipids was 1:1, w/w), and the obtained lyophilized liposomes showed good redispersibility as well as homogeneity. The mean diameters of TFu-loaded liposomes changed to (438.9±10.7) nm from 400nm after lyophilization, and the polydispersity was 0.651, zeta potential was -4.3mV, the drug entrapment efficiency was (75.15±3.90)%, the drug loading was (7.52±0.24)%, respectively. The formation of TFu-loaded liposomes was validated by DSC, which indicated that TFu successfully encapsuled into liposomes. Result of the preliminary stability experiments indicated that thelyophilized TFu-loaded liposomes were almost intact at 4℃ for 9 month.4. Pharmacokinetics, tissue distribution and bioavailabilities of TFu-loadedliposomes with different sizes after intravenous and oral administrationThe liposomes size may be a key factor on the enhancement of the oral uptake of encapsulated drugs. The pharmacokinetics characteristics and bioavailability of liposomes with different sizes (530, 400, 320 and 180nm) as well as the tissue distribution of TFu-loaded liposomes (400nm) were investigated after oral administration to mice. The relative bioavailabilities of TFu liposomes with sizes of 530nm, 400nm, 320nm and180nm were 160.5%, 198.9%, 221.6% and 260.3%, respectively. The pharmacokinetics study indicated that TFu-loaded liposomes with different sizes all resulted in higher bioavailabilities than the TFu suspension after oral administration, and gastrointestinal absorption increased with the reduction of liposome sizes. The tissue distribution showed liposomal formulation also enhanced drug uptake in liver and spleen besides in plasma.The pharmacokinetic behavior and tissue distribution characteristic of a drug may be completely altered by entrapping the drug into liposomes with sizes of 530nm, 400nm and 180nm. After i.v. administrated to mice, larger TFu-loaded liposomes with 530nm and 400nm sizes both showed passive hepatic and splenic targeting properties and lower cardial and renal accumulations (p<0.01; p<0.01) compared with TFu 50% alcohol solution; While smaller size liposomes with 180nm size reduced the uptake of TFu in reticuloendothelial system (RES) organs, enhanced drug concentration in plasma, bioavailability and retention time significantly, thereby exhibited a signification long-circulation property.In a word, oral and injectable TFu-loaded liposomes were promising anticancer formulations for the improved bioavailability, and the different sizes of TFu-loaded liposomes might offer more convenience and practical preparations to meet the different clinical therapeutic needs and provide targeted chemical therapy effects. 5. Oral absorption mechanism of TFu-loaded liposomesThe influence of tween-80, blank liposomes and dinitrophenol on the oral absorption of TFu-loaded liposomes were investigated by in vivo pharmacokinetic studies. TFu solid lipid dispersions were oral administrated to mice to explore the differences in oral uptake between solid dispersions and liposomes. Further, everted intestinal sac was applied to investigate oral absorption mechanism of TFu-loaded liposomes.Compared with the control group, drug uptake did not decrease (p>0.05) when blank liposmes were mixed with TFu-loaded liposomes, while the energy inhibitor (dinitrophenol) and surfactant (tween-80) improved the oral absorption of TFu. The transport of TFu-loaded liposomes across small intestine might be a passive diffusion process which was carrier-independent, energy-independent. Besides, the everted intestinal sac showed the diffusion process was dose-dependent, and fitted with Fick's diffusion law.When TFu-loaded liposomes were incubated in gastric or intestinal contents of mice, the drug entrapment efficiency reduced by 17% and 5% compared with in PBS7.4, respectively, but the EE were both above 50%. The results indicated TFu-loaded liposomes were instabe in gastrointestinal tract and some amounts of them were degraded.The everted intestinal sac method was applied to investigate the permeability of TFu-loaded liposomes with different sizes (530, 400, 320 and 180nm). The content of encapsuled drug inner mice intestine segment was almost zero though the concentration of free TFu increased all the way, while encapsulation efficiencies of liposomes with different sizes all decreased as the time going on as well as the free TFu concentration. Differences (p<0.01) existed among the TFu-loaded liposomes with different sizes, smaller liposomes (180nm) presented a higher intestinal penetration effect.These results revealed that it might be the TFu molecule itself not the intact TFu-loaded liposomes could cross the intestinal wall.There were no significant differences in oral absorption between TFu solid lipid dispersions and TFu-loaded liposomes after oral administrated to mice. Liposomal bilayer might not have superiority for protecting drug in gastrointestinal tract, and accordingly enhanced bioavailabilities significantly compared to the TFu solid lipid dispersions. However, solid lipid dispersions showed bad regularity in vitro release, the changes for accumulative release exceeded to 20%, and the clinical application might be limited by its ageing properties. Compared with solid dispersions, liposomes were more stable and the benefit of liposomes over free drug is clinically well-established.In this study, a poorly water soluble prodrug TFu was successfully incorporated into liposomes for the aim to apply to oral and intravenous administration. TFu-loaded liposomes had a higher entrapment efficiency using the film dispersion-homogenization technique and lyophilized TFu-loaded liposomes could keep stable at least within 9 months. The liposomes did enchance the gastrointestinal absorption of TFu by oral administration, and gastrointestinal absorption increased with the reduction of liposome sizes. After i.v. administrated to mice, larger TFu-loaded liposomes with 530nm and 400nm sizes both showed higher hepatic and splenic targeting properties and lower cardial and renal accumulations compared with TFu 50% alcohol solution; while smaller size liposomes with 180nm size enhanced drug plasma concentration, bioavailability and retention time significantly. While smaller size liposomes with 180nm size enhanced drug plasma concentration and prolonged the circulation time, thereby exhibited a good long circulating effect. Injectable TFu-loaded liposomes with different sizes might have different clinical utilizations concerning different treatment goals. These results indicated that liposomes could offer a promising way to improve the bioavailability and the therapeutic efficacy of poorly soluble lipophilic drugs. Further, the studies on the oral enhancing absorption mechanism of TFu-loaded liposomes provided scientific experimental and theoretical basis for developing clinical practical oral liposomes formulation.