Study On Liposomes Modified With PEtOz-CHEMS Lipid-Derivatives

Poly(2-ethyl-oxazoline)-cholesterol hemisuccinate (PEtOz-CHEMS) was synthesized from PEtOz, With good hydrophilicity, flexibility, biodegradability and pH-sensitivity. As a pH-sensitive block copolymers, on the one hand, PEtOz-CHEMS could increase the stability of liposomes and prolong the circulation time. On the other hand, PEtOz-CHEMS could provide pH-sensitivity of liposomes and made it release drug on the low pH position in vivo, and thus an ideal drug delivery carriers have been obtained. In this study, we took Superoxide dismutase (SOD)mimics as a model drug and made use of PEtOz-CHEMS to build long circulating pH-sensitive liposomes. And we have a systematic investigation on them.A vitro analysis method was established in the preformulation studies. The concentration of SOD mimics was determined by UV-Vis. We have reviewed the solubility of SOD mimics in different solvents, and the physical and chemical properties of them have been studied. The results show that the SOD mimic is relatively stable, which is almost insoluble in the water but soluble in a small number of organic solvents and it is also found that UV-Vis can be used as a efficient method to determine the SOD mimics concentration.We Selected Cholesterol, Succinic anhydride and PEtOz as starting raw material to esterification reaction and eventually the long-circulating pH-sensitive material PEtOz-CHEMS was synthesized. The synthetic products were identified by UV, IR and 1H-NMR. And the critical mical concentration (CMC) was determined by maximum bubble pressure method.The SOD mimics liposomes were prepared by film-disersion method. The entrapment efficiency of SOD mimics in liposomes was determined by using the Sepadex G-50 mini-column centrifugation method. Via species of phospholipid, durg-lipid ratio, addition of cholesterol and hydration mediator et al as indexes. The liposomes with average diameter of 111.8 nm. The suitable formulation was got with the entrapment efficientdy above 91% with the preparation method.Using PEtOz-CHEMS modified liposomes, we determined the calcein liposomes'release in vitro by dialysis metheod in pH 7.4 buffer solution within 18 hour. The general release liposome to (51.05±1.33)% at 18 hour, whereas the common general can release liposome to (76.5±1.34)% at 15 hour. The results show that the stability of the liposomes modified by PEtOz-CHEMS were greatly improved. We have used two methods to verify the acid sensitivity of liposomes. The release rate of the calcein liposomes in vitro was (94.6±1.33)%, (88±1.35)%, (80±1.49)% in different pH buffer solution (4.4,5.4,6.4) within 18 h which is modified by PEtOz-CHEMS. The release rate were much higher when the pH is 7.4. The liposomes, which is modifid by PEtOz-CHEMS, incubated with rabbit red blood cells. The results show that the release amount of them in pH-sensitive liposomes group have close to the positive control group when the pH is 6 and incubation time 2.5 hour. It have proved that the pH-sensitive liposomes under acidic conditions could occur degree of integration with the red cell membrane, and then caused the release of hemoglobin. These results proved that the liposomes modified by PEtOz-CHEMS have good acid sensitivity.To improve the stability of SOD mimics liposomes, in the experiment, we adopted uniform design method to select the kinds and adscititious of the dry protection reagent and refined the freeze-drying process. Finally, the best freeze-drying protectants were prepared. A mixture of 14% lactose,1% mannitol and8% trehalose as protective agents have caused optimal appearance full, smooth, exquisite and without crystallization white pie of SOD mimics free-dried liposomes.The activity and safety of SOD mimics liposomes in vitro was discussed and evaluated. The NBT riboflavin photoreduction method was taken to examine the activety of the liposomes. The experiment proved that the activity of the SOD mimics did not change after it was encapsulated in the liposomes, so it would not impact on the further administration. Based on the conventional test tube method in vitro and combined with the spectrophotometry to investigate hemolytic of the different prescription of samples, the results show that the SOD mimic liposomes have no hemolytic which has provided the basis of ensuring the drug safety.