CHARACTERIZATION AND OPTIMIZATION OF PEPTIDE ENCAPSULATION IN PEGYLATED LIPOSOMES

Purpose: Print 3G is a peptidic antagonist of oncoprotein involved in breast cancer, containing 25 amino acids. The purpose of this work is to study the peptide encapsulation into PEGylated liposomes. Two formulations composed of SPC:CHOL:mPEG-750-DSPE (47:47:6) or SPC:CHOL:mPEG-2000-DSPE (47:47:6) were investigated.

Methods: Unilamellar vesicles containing either mPEG750 or mPEG2000 were prepared by hydration of lipid films method. Unfortunately, a loss of Print 3G was observed during the different steps of this manufacturing technique giving rise to encapsulation efficiencies close to 0 %. Thus, the freeze-thawing method was used to enhance the amount of Print 3G encapsulated into blank liposomes prepared using the above procedure. Because many factors may influence the peptide entrapment into the vesicles (number of freeze-thawing cycles, lipid concentration, peptide concentration, mixing time and liposome composition), a design of experiment was performed (for the screening, a Plackett and Burman plan; for the optimization, a central composite design).

Results: The encapsulation efficiencies obtained by the freeze-thawing method in standard conditions, varied between 17.26 ± 0.46 % (n=3) for liposomes containing mPEG750 and 26.20 ± 7.98 % (n=3) for those comprising mPEG2000. Among the different considered factors, the screening permitted to identify two factors having a positive and significant influence on the encapsulation efficiencies: the number of freeze-thawing cycles and the lipid concentration, while the presence of mPEG2000 or mPEG750 had a relatively weak effect on the encapsulation. Concerning the peptide concentration and the mixing time, no influence was revealed. For the second part of the DOE, the positive factors were optimized for the liposomes containing mPEG2000. The obtained results for liposomes containing mPEG2000 revealed a theoretical optimum at 64.75 ± 3.55 % when 11 freeze-thawing cycles were applied and for the following lipid concentrations: 36.1 mM SPC, 36.1 mM CHOL and 4mM mPEG-2000-DSPE. The experimental results showed an encapsulation efficiency of 62.68 ± 2.93 %.

Conclusion: Changing the manufacturing technique permitted a significant encapsulation of Print 3G into liposomes. The DOE led to a significant improvement of encapsulation efficiencies for the liposomes containing mPEG2000. Thereafter, an optimization design for liposomes containing mPEG750 will be started.

Acknowledgements: This work was supported by the Ministry of the Walloon Region.