Increasing the Entrapment of Protein-Loaded Liposomes with a Modified Freeze–Thaw Technique: A Preliminary Experimental Study

Abstract

The objective of this study was to investigate the possibility of increasing the entrapment of protein-loaded liposomes with a modified freeze–thaw technique. Blank liposomes were prepared by ethanol injection method. Then recombinant human growth hormone (rhGH) was added to blank liposome suspension. rhGH encapsulation efficiency was enhanced by modified freeze–thaw technique. We separated each step and studied the effect of each parameter on encapsulation: incubation temperature (water bath from 0 to 15°C), incubation duration (from 5 to 90 min), number of freeze–thaw (from 0 to 4 cycle), and the ratio between rhGH and phospholipids (from 1:5 to 1:10). The effect of cryoprotectants on the encapsulation and particle size distribution was finally examined. rhGH encapsulation efficiency was determined by Bradford's dye-binding assay. Morphology and size distribution of rhGH liposomes were also observed. The optimum parameters for rhGH encapsulation were incubation temperature of 5°C, incubation duration of 40 min, and three to four cycles of freeze–thaw. The ratio between rhGH and phospholipids did not affect the encapsulation percentage. Trehalose exhibited the highest integrated value among the cryoprotectants investigated. From the results, this study demonstrates the suitability of the modified freeze–thaw technique for obtaining rhGH liposomes with high entrapment.