The Preparation And Studies On Iohexol Liposomes In Vitro And In Vivo

With the development of medical image and the requirement of disease diagnosis, contrast agent has been applied in clinic more and more, the safty of which has been paid greater attention by clinicians and patients.Iohexol is a water soluble X-CT contrast agent, which was produced by Metrizamide in Norwegian in 1980s and thought to be one of the best non-ionic X-CT contrast agent. However, the application of Iohexol in clinic has been limited because of its drawbacks such as large dosage, short roentgenopaque time, vascular poison, nephrotoxicity and neurotoxicity.In the present study , Iohexol was taken as the model drug to prepare liposomes. The stability of drug has been improved due to the entrapment and targeting of liposomes, and the dosage has been reduced, accordingly its side effects have been decreased. Thus the study has a very high scientific and practical value. The main results were as follows:1. The entrapment efficiency and drug concentration were determined by ultraviolet spectrophotometry. The sephadex G-50 column method and the ultracentrifugation method were compared to separate the free drug and Iohexol liposomes. There was a good linearity of the drug concentration within the range of 2.5-25.0μg/ml and the linear equation was A = 0.0312C + 0.0099 (r=0.9999). The recovery rate was between 95% and 102% and the RSD (intra- and inter-day) was less than 2%.2. The preparation of liposomes was investigated by film dispersion method, film ultrasonic method, ether injection method and reverse phase evaporation method, respectively. The entrapment efficiency of liposomes prepared by different methods was 22.5±3.5%,8.9±2.4%,10.9±6.6% and 65.9±4.9%, respectively. Ultimately, reverse phase evaporation method was selected to prepare Iohexol liposomes. The entrapment efficiency was taken as a criterion to evaluate the effects of the ratio of lipids, the volume of organic phase and water phase, drug concentration, sonicating time, sonicating intensity and water bath temperature on the properties of Iohexol liposomes. As a result, the weight ratio of soya lecithin to cholesterol, weight ratio of drug to lipids, volume ratio of ether to phosphate buffer were proved to have great effects on the properties of liposomes. Orthogomal design was applied to optimize the formulation. The Iohexol liposomes were spherical vesicles with uniform sizes. The mean diameter of blank liposomes and Iohexol liposomes were (166.2±28.6) nm and (189.1±49.6) nm, and the polydispersity were 0.461 and 0.262, respectively.The drug contents were (99.4%±0.95), the zeta potential was (-34.0±1.65)mv. The drug loading and entrapment efficiency were (38.83%±1.05) and(77.66%±2.10, respectively, pH was (6.42±0.12).The results indicated thatthe Iohexol liposomes had a higher entrapment efficiency, and the particlesize as well as charge were in accord with the targeting demands whichreached the expectant objective.3. The in vitro release profiles of Iohexol solution could be described by zero order dynamic model and could be expressed by the following equation Q%=167.38t+17.284 (r=0.9974). The in vitro release behavior of Iohexol from Iohexol liposomes could be described by first order dynamic model and expressed by the following equation Ln(100-Q)=-0.17t+4.5974 (r=0.9954).4. The stability test of negatively charged Iohexol liposomes showed that the entrapment efficiency of Iohexol liposomes were 78.4%,77.2%,76.3%,73.0% and 71.8% at 4℃for 1 day,7 days,15 days and 30 days, respectively, which indicated the Iohexol liposomes had a good stability.The physical stability of negatively charged Iohexol liposomes was shown as follows: Firstly, Iohexol liposomes showed unstable under heating conditions, the entrapment efficiency of Iohexol liposomes were 75.13% and 21.25% before and after heating, respectively; Secondly, under the storage condition of 25℃for two months, the drug entrapment efficiency decreased significantly, from 74.17% to 46.13%, and the particle size increased obviously, from 298.37 nm to 701.25 nm. However, in the case of 4℃for four months, the drug entrapment efficiency decreased slightly, from 74.17% to 63.14% , and the particle size increased slightly, from 298.37 nm to 349.8 nm, which indicated that Iohexol liposomes needed to be stored under cold conditions. Thirdly, the drug entrapment efficiency was affected by the dilution multiple: the entrapment efficiency decreased with increasing the dilution multiple. Moreover, the drug entrapment efficiency was found to be affected by osmotic pressure according to the comparative study between water and normal saline (N.S.), while the iso-osmia dissolvent influenced the drug entrapment efficiency slightly.The chemical stability of Iohexol liposomes was shown as follows: First, adding V_E could prevent Iohexol liposomes from oxidizing, accordingly improving its stability; Second, ⁶⁰Co radiation had less effect on the stability of Iohexol liposomes.5. The comparative CT experiment between Iohexol injection solution and Iohexol liposomes was carried on rabbits. After 0 min of the liver CT scanning, the CT signal for Iohexol injection solution appeared, afterwards, the CT signal decreased obviously; After the Iohexol liposomes were administrated to rabbits, the maximum CT signal appeared in 10 min, and the intension of which was much higher than that of the Iohexol injection. The CT signal at 30 min after administration of liposomes was still above the CT signal after the Iohexol injection solution injected immediately. When the dosage of Iohexol liposomes was cut down to 1/8, the enhancement effect on CT signal was still observed obviously. The results of the CT scanning in the muscle and brain of rabbits were similar to that in liver after administration of Iohexol injection solution and Iohexol liposomes.The Iohexol liposomes showed a good targeting and could achieve the same therapeutic effects with Iohexol injection solution while decreasing the dosage and the toxicity of the contrast medium.