Liposomes Interiorly Thickened By Gelatin As Drug Delivery Carrier

Liposomes have been used extensively in the field of drug delivery owing to their intrinsic advantages. However, the potential application of liposomes is still challenged by their inherent physical and chemical instabilities in aqueous dispersions, which can result in drug loss, vesicle aggregation/fusion, and drug phasing out. Freeze-drying is an effective approach to prepare solid proliposomal formulations overcoming stability problems. However, freeze-drying procedures might strongly affect liposome structure and result in difficult reconstitution. In the present study, our hypothesis is to incorporate some kinds of support in the liposome interior to reserve the liposomal structure to survive the freeze-drying cycle. Microscale interior gelatin-thickened liposomes (GTLs) were first prepared for ease of verification of the phenomenon of interior gelation and in vitro characterization of GTLs. On this basis, potential application of GTLs was further explored by using of nanoscale gelatin-thickened liposomes containing bile salts (D-GTLs) as vehicles to enhance the oral bioavailability of the poorly water-soluble and poorly permeable drug.The thin-film dispersion method was used to prepare GTLs and the morphology was characterized by transmission electron microscope (TEM), which revealed no obvious difference between GTLs and CLs. The existence of interior gel was further identified under the fluorescence microscope by addition of Triton X-100 into the gelated 5(6)-carboxyfluorescein (CF) liposomes. The fluorescence was reserved because of the retaining of the gel, where as non-thickened liposomes lost fluorescence due to complete disruption of the liposomal structure. The gelatination temperature (Tgel) of gelatin solution was determined by the stirring method and the interior gelatination temperature of GTLs (Tgel-in) was determined by CF release from GTLs. The results showed that Tgel-in was slightly lower as compared with Tgei.Membrane fluidity of GTLs was assessed by measuring fluorescence polarization of diphenylhexatriene (DPH) and the results demonstrated that membrane fluidity was decreased by interior gelatination of GTLs.Paclitaxel (PTX)-loaded freezed-dried GTLs were prepared by the thin-film dispersion method followed by freeze-drying. SEM was used to observe the morphology of freeze-dried liposomes. GTLs kept the structure of liposomes after lyophilization and not any PTX crystal was observed. However, recrystallization and phasing out of PTX from the surface of particles were clearly observed in CLs under SEM. The entrapment efficiency of freezing-dried liposomes was compared between GTLs and CLs. It was shown that the entrapment efficiency of freeze-dryied CLs decreased to 18.1% after reconstitution, and the result of CLs with lyoprotectant presenting in both interior and exterior of the vesicles was similar. However, the encapsulation efficiency of GTLs was still above 84.9% after reconstitution regardless of the interior concentration of gelatin.Cyclosporine A (CyA)-loaded freezed-dried GTLs containing a bile salt were prepared by thin-film dispersion, homogenization and freeze-drying methods. TEM was carried out to describe the morphology of freeze-dried liposomes after reconstitution. Aggregation of vesicles could only observed in freeze-dried DLs after rehydration, but not in freeze-dried D-GTLs under TEM. The particle size and PI of freeze-dried D-GTLs exhibited no obvious change after rehydration, whereas the particle size and PI of DLs significantly increased from about 85 nm to 400 nm, and 0.1 to 0.4, respectively.Pharmacokinetic study was carried out in rats after oral administration of CyA-D-GTLs, CyA-DLs, CyA-CLs and microemulsion-based Sandimmun Neoral?. The results showed improved absorption of CyA in D-GTLs as compared to CLs and Sandimmun Neoral?. The relative oral bioavailability of CyA-D-GTLs and CyA-CLs was 118.4% and 98.6%, respectively, with Sandimmun Neoral? as the reference. There was no significant difference of pharmacokinetics parameters compared between CyA-D-GTLs and CyA-DLs.Pharmacokinetic study was further carried out in Beagle dogs after oral administration of CyA-pro-D-GTLs, CyA-pro-DLs, CyA-D-GTLs and Sandimmun Neoral?. The results showed improved absorption of CyA in proD-GTLs as compared to pro-DLs, D-GTLs and Sandimmun Neoral?. The relative oral bioavailability of CyA-pro-D-GTLs, CyA-pro-DLs and CyA-D-GTLs was 165.2%,123.5 and 130.1%, respectively, with Sandimmun Neoral? as the reference. The Tmax, Cmax and AUC0-t of CyA-pro-D-GTLs were all significantly higher than that of CyA-pro-DLs, which confirmed the superiority of interior gelatin-thickened liposomes on solidification of liposomes containing bile salts.The jugular vein- mesenteric lymph duct cannulated conscious rat model was employed to investigate the intestinal lymphatic transport of CyA. The extent of lymphatic transport of CyA-D-GTLs was 0.7% of the dose administered, which was higher than that of CyA-CLs and lower than that of Sandimmun Neoral?. The results indicated that the potential mechanism of enhanced oral absorption of CyA from CyA-D-GTLs compared with CyA-CLs was that intact liposomal vesicles may promote the uptake by M-cells in the Peyer’s patches and increasing the absorption through the lymphatic pathway. However, several mechanisms may contribute to the superiority of CyA-D-GTLs to Sandimmun Neoral? and need further elucidation in vivo.Gelatin was successfully used to thicken the interior of liposomes. The thickened interior of the liposomes acted as interior support during freeze-drying and improved stability was achieved for liposomes. It is concluded that liposomal interior thickening with gelatin holds great potential in preparation solid liposomal formulations.