Preparation of liposomes and Encapsulation of Bioactives Using Supercritical Carbon Dioxide

• Author / Creator

  Zhao, Lisha

• Liposomes are versatile vesicles of a phospholipid bilayer, which offer great protection for active compounds. Encapsulated compounds can be protected from the unfavorable external conditions so that their chemical stability and bioavailability can be greatly enhanced. The supercritical carbon dioxide (SC-CO2) technique has drawn great attention over the traditional preparation methods with unique advantages. To yield liposomes with improved characteristics, an improved SC-CO2 method, which combined the processing advantages of two previously reported methods was developed for liposome preparation, which were then loaded with bioactives in this PhD research. First, the effectiveness and fundamentals of a previously reported SC-CO2 method was studied by equilibrating a soy lecithin aqueous suspension with high pressure CO2. Unloaded liposomes were then formed via the depressurization from the supercritical phase. The relative volumetric expansion of water upon equilibrium with CO2 was 14.36%, 12.28% and 10.24% at 40°C, 50°C and 60°C, respectively. Liposomes had a particle size of 523±2 nm and a polydispersity index (PdI) of 0.226±0.01. Increased depressurization rate led to an improved homogeneity of vesicles and liposomes could be stored for up to 4 weeks with limited size change (below 5%). SC-CO2 method displayed advantages of high stability and homogeneity for liposome preparation over the conventional thin film hydration (TFH) method. Second, the improved SC-CO2 method was developed, where unloaded liposomes were formed via the depressurization from the liquid phase following two depressurization protocols: (I) depressurization at a constant rate and (II) depressurization at a constant rate and pressure. The effects of processing and compositional parameters on the characteristics of liposomes were studied. Liposomes prepared by the improved SC-CO2 method had a particle size of 265±4 nm and 214±3 nm and PdI of 0.520±0.02 and 0.417±0.001 for protocols (I) and (II), respectively. Protocol (II) yielded smaller and more uniform particles. Liposomes were stable for up to 8 and 6 weeks for protocols (I) and (II), respectively. Elevated phospholipid level resulted in smaller particle size with enhanced uniformity. Fatty acids with longer chain length in pure phospholipids resulted in a larger particle size while increased degree of unsaturation in the fatty acyl chains increased the asymmetry of vesicles. The improved supercritical method was more effective in liposome preparation than the TFH method for a reduced size and enhanced stability. Next, lutein (hydrophobic) was encapsulated into liposomes by the improved SC-CO2 method via the depressurization protocol (II). The effects of processing parameters and compositional parameters on the characteristics of liposomes were studied. Liposomes had a particle size of 155±1 nm, an encapsulation efficiency (EE) of 97.0±0.8% and a zeta potential of -61.7±0.6 mV. The higher pressure and depressurization rate promoted enhanced lutein encapsulation efficiency whereas the lutein concentration could significantly influence the morphology of vesicles along with size distribution and EE. Finally, anthocyanin (hydrophilic) was encapsulated into liposomes by the improved SC-CO2 method via the depressurization protocol (II). The effects of processing parameters and compositional parameters on the characteristics of liposomes were studied. The in vitro release of bioactives from liposomes was also investigated. Liposomes obtained had a particle size of 160±2 nm, EE of 52.2±2.1% and zeta potential of -41.3±1.2 mV. Increased pressure and depressurization rate resulted in reduced particle size and enhanced uniformity while the high temperature increased the fluidity of bilayer membrane. Elevated anthocyanin level increased the particle size with reduced uniformity while the elevated cholesterol concentration reduced the zeta potential of liposomes. Liposomes were stable for up to 3 weeks with a unimodal narrow size distribution. The release of anthocyanin from the liposomes was slow with up to 35.9% in the simulated gastric fluid while it was rapid in the simulated intestinal fluid due to the liposome degradation by pancreatin. In summary, the improved SC-CO2 process was effective to finely disperse the amphiphiles mixed with hydrophobic or hydrophilic bioactives suspended in an aqueous system into uniform nano/micro particles. The processing and compositional factors can be fine tuned to modulate the physicochemical properties of the liposomes obtained, depending on the targeted applications. This novel process is advantageous for potential scale-up of the production of micro/nano particles with desirable characteristics and shows great promise to encapsulate a variety of valuable bioactives for food, nutraceutical and medical applications.

• Subjects / Keywords

  • Bioactive
  • Nanoparticles
  • Supercritical carbon dioxide
  • Encapsulation
  • Liposomes

• Graduation date

  Spring 2017

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/R3H12VK9X

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.