STUDY OF PEGYLATED LIPOSOMES CRYOPROTECTION IN TERMS OF RETENTION EFFICIENCY

Purpose: Print 3G, a peptidic antagonist of oncoproteins involved in breast cancer, was encapsulated in PEGylated liposomes. Seeing its temperature instability, this peptide must be stored at – 20°C. In this study, the cryoprotection of PEGylated liposomes was investigated to ensure their stability in terms of size and retention capacity when frozen. Calcein, a self-quenching fluorescent marker encapsulated in liposomes, was used as a tracer of liposome integrity. The release of calcein was studied before and after freezing, with or without cryoprotectants at several concentrations. Methods: Unilamellar vesicles made of SPC:CHOL:mPEG-750-DSPE (47:47:6) or SPC:CHOL:mPEG-2000-DSPE (47:47:6) were prepared by the film evaporation method. Size of the liposomes dispersions containing a cryoprotectant (sucrose, trehalose or lysine) at various concentrations were measured by photon correlation spectroscopy before and after freezing. From these results, two cryoprotectants (at the best molar ratio of sugar-to-lipid) were chosen for future experiments with calcein. Its release from the inner cavity of liposomes was measured fluorometrically before and after freezing, with or without cryoprotectant. The percentage of calcein retained was determined at excitation and emission wavelengths of 490 and 520 nm, respectively. The liposomes were prepared with cryoprotectant either only outside the vesicles or both inside and outside. Results: In regards to the results obtained with PCS, sucrose and trehalose (molar ratio of sugar-to-lipid = 4) were selected for calcein experiments. The best results in terms of retention efficiency were obtained using cryoprotectant inside and outside the vesicles. Liposomes containing mPEG-750-DSPE show the best retention efficiency after freezing with trehalose (75.42 ± 2.39 %, n=3) in comparison with sucrose (62.16 ± 4.34 %, n=3). Liposomes containing mPEG-2000-DSPE doesn’t show significant difference (T-Test) between trehalose (84.27 ± 5.05%, n=3) and sucrose (83.38 ± 3.18 %, n=3). In the future experiments, these results will be compared with experiments of frozen liposomes encapsulating Print 3G. Acknowledgements: This work was supported by the Ministry of the Walloon Region.Peptides antagonistes d'oncoprotéines pour une thérapeutique du cancer du sei

FORMULATION OF PEPTIDE-LOADED LIPOSOMES FOR INTRAVENOUS DELIVERY: OPTIMIZATION OF THE ENCAPSULATION EFFICIENCY

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 composed of SPC:CHOL:mPEG-2000-DSPE (47:47:6). Methods: Loaded unilamellar vesicles were prepared by hydration of lipid film method. Unfortunately, a loss of Print 3G was observed during the different steps of this manufacturing technique giving rise to encapsulation efficiencies (EE) 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 (NC), lipid concentration (LC), peptide concentration (PC) and mixing time (MT)), a design of experiment (DOE) was performed (for the screening, a Plackett and Burman plan; for the optimization, a central composite design). EE were calculated in terms of quantity of peptide loaded in liposomes as a function of quantity operated (EEp) or quantity of lipids (EEl). Results: The EE obtained by the freeze-thawing method in standard conditions (Katanasaka, Ida et al., 2008; Maeda, Bharate et al., 2008), amounted to 26.20 ± 7.98 %, n=3 (EEp) and to 0.26 ± 0.074 % (EEl). Among the different considered parameters, the screening permitted to identify two factors having a positive and significant influence on the EE: NC and LC. Concerning the PC and MT, no influence was revealed. For the second part of the DOE, the positive factors were optimized and obtained results revealed a theoretical optimum at 64.75 ± 3.55 % when 11 freeze-thawing cycles were applied (NC) and for the following LC: 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 %. Photon correlation spectroscopy and freeze-fracture electron microscopy were also realized to examine the liposome integrity, before and after the freeze-thawing cycles. Conclusion: Changing the manufacturing technique permitted a significant encapsulation of Print 3G into liposomes. The DOE led to a significant improvement of encapsulation efficiencies. References: (1) Katanasaka,Y., Ida,T., Asai,T., Maeda,N., Oku,N., 2008. Effective delivery of an angiogenesis inhibitor by neovessel-targeted liposomes. International Journal of Pharmaceutics, 360, 219-224. (2) Maeda,H., Bharate,G.Y., Daruwalla,J., 2008. Polymeric drugs for efficient tumor-targeted drug delivery based on EPR effect. Eur. J Pharm Biopharm. Acknowledgements: This work was supported by the Ministry of the Walloon Region.Peptides antagonistes d'oncoprotéines pour une thérapeutique du cancer du sei

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.Peptides antagonistes d'oncoprotéines pour une thérapeutique du cancer du sei

CARACTERISATION ET OPTIMISATION DE L’ENCAPSULATION D’UN PEPTIDE ANTAGONISTE D’ONCOPROTEINE AU SEIN DE LIPOSOMES PEGYLES

Introduction : Print 3G est un peptide antagoniste d’une oncoprotéine impliquée dans le cancer du sein, composé de 25 acides aminés. Le but de ce travail est d’étudier l’encapsulation de ce peptide au sein de liposomes pegylés. Deux types de formulations de liposomes ont été étudiés, le premier contenant des mPEG750-DSPE et le second des mPEG2000-DSPE. Méthode : Des liposomes unilamellaires composés de SPC:CHOL:mPEG750-DSPE ou de SPC:CHOL:mPEG2000-DSPE (47:47:6) encapsulant le peptide ont été préparés par la technique d’évaporation du film. L’observation d’une adsorption conséquente de Print 3G sur les instruments utilisés a conduit à l’utilisation de la méthode de fabrication par cycles gel-dégel. Etant donné que plusieurs facteurs peuvent influencer l’encapsulation du peptide à l’intérieur des liposomes, une planification expérimentale a été engagée (pour le screening, un plan de Plackett et Burman et pour l’optimisation, un plan composite faces centrées). Résultats : Par la technique des cycles gel-dégel généralement décrite dans la littérature(1; 2), les taux d’encapsulation obtenus étaient de 17,26 ± 0,46 % (n=3) pour les liposomes contenant des mPEG750 et de 26,20 ± 7,98 % (n=3) pour ceux contenant des mPEG2000. Le plan de screening a permis de dégager deux facteurs ayant une influence positive sur l’encapsulation : la concentration en lipides et le nombre de cycles gel-dégel. Ceux-ci ont été optimisés pour chaque type de mPEG. Les résultats obtenus pour les mPEG2000 mettent en évidence un optimum théorique de 64,75% pour un nombre de cycles égal à 11 et pour une concentration en lipides SPC:CHOL:mPEG2000-DSPE de 36.1mM:36.1mM:4mM. Les résultats expérimentaux ont permis d’obtenir un taux d’encapsulation de 62,68 ± 2,93 %. Conclusion et perspectives : Le changement de technique de fabrication a permis l’encapsulation significative du peptide à l’intérieur des liposomes. La planification expérimentale a ensuite conduit à l’amélioration significative des taux d’encapsulation pour les liposomes contenant des mPEG2000. Par la suite, un plan d’optimisation pour les liposomes contenant des mPEG750 sera mis en œuvre. Remerciements: Ces recherches ont été réalisées avec le soutien de la Région Wallonne. Références: 1. Y.Katanasaka, T.Ida, T.Asai, N.Maeda, and N.Oku. Effective delivery of an angiogenesis inhibitor by neovessel-targeted liposomes. International Journal of Pharmaceutics, 360 (1-2):219-224 (2008). 2. G.A.Ramaldes, J.-R.Deverre, J.-M.Grognet, F.Puisieux, and E.Fattal. Use of an enzyme immunoassay for the evaluation of entrapment efficiency and in vitro stability in intestinal fluids of liposomal bovine serum albumin. International Journal of Pharmaceutics, 143 (1):1-11 (1996).Peptides antagonistes d'oncoprotéines pour une thérapeutique du cancer du sei

Cellular uptake of long-circulating pH-sensitive liposomes: evaluation of the liposome and its encapsulated material penetration in cancer cells

Print 3G, a peptidic antagonist of oncoprotein involved in breast cancer, could reduce the angiogenic development of breast tumors, leading to tumor dormancy. The necessity of intravenous administration of Print 3G led to the development of long-circulating liposomes as drug carriers. Pegylated liposomes, too large to be collected by fenestrated organs, accumulate passively in solid tumors thanks to the EPR effect. The strategy was to combine the protective properties of PEG with the transfection properties of pH-sensitive lipids which could promote the uptake of liposomes by cells and avoid lysosomal sequestration and degradation of entrapped materials such as peptides. In this study, we compare two formulations in terms of cellular uptake using confocal microscopy. The first one is composed of SPC:CHOL:mPEG-750-DSPE (47:47:6), used as "standard" liposomes, and the second one of DOPE:CHEMS:CHOL:mPEG750-DSPE (43:21:30:6), used as pH-sensitive liposomes. First, we evaluated the penetration of an encapsulated model molecule, calcein, in Hs578t human breast cancer epithelial cells. When calcein was encapsulated in standard liposomes, its penetration was effective only in few cells. On the contrary, a majority of cells were fluorescent when calcein-loaded pH-sensitive liposomes were applied for 3 hours on cells. Secondly, we studied the penetration of liposomes themselves in Hs578t cells using 25-[(nitrobenzoxadiazolyl)methylamino]nor-cholesterol (NBD-CHOL) as a fluorescent marker of the phospholipid membrane. The obtained results were comparable to those obtained with calcein: a higher penetration of liposome was observed for pH-sensitive liposomes. Finally, the cellular uptake of liposomes using both NBD-CHOL and rhodamine encapsulated in the inner cavity of vesicles was evaluated with Hs578t cells and compared with WI26 human diploid lung fibroblast cells. Thanks to this experiment, we could follow simultaneously the cell distribution of the encapsulated material and of the liposome itself. Confocal pictures obtained with pH-sensitive liposomes on both WI26 and Hs578t cells allow us to visualize the co-localized red and green colors of rhodamine and NBD-CHOL, with a higher concentrated area near the nucleus. In comparison with "standard" liposomes, we observed a higher penetration of the encapsulated material and of the liposome itself in breast cancer cells. Moreover, we visualized a colocalization near the nucleus of liposomes components. Concerning results obtained with fibroblastic cells, there was no difference in terms of cellular uptake between the two formulations. In perspective, we would like to compare these results, obtained with model molecules, with experiments performed with biotinylated Print 3G to assess its cellular distribution. Moreover, it would be interesting to correlate results obtained with confocal microscopy with a possible increase of the peptide efficacy against cancer cells when it is encapsulated in long-circulating pH-sensitive liposomes.Peptides antagonistes d'oncoprotéines pour une thérapeutique du cancer du sei

PEPTIDE-LOADED LIPOSOMES AGAINST BREAST CANCER: EFFECTIVE PENETRATION IN CELLS OF LONG CIRCULATING pH-SENSITIVE VESICLES

Purpose: Print3G, a peptidic antagonist of oncoprotein involved in breast cancer, could reduce the angiogenic development of breast tumors. The necessity of intravenous administration of Print3G led to the development of liposomes as drug carriers, combining the protective properties of PEG with the transfection properties of pH-sensitive lipids. The purpose of this work is to compare pegylated pH-sensitive liposomes with a classical formulation of long-circulating liposomes in terms of cellular uptake. Methods: Classical liposomes (SPC:CHOL:mPEG-750-DSPE (47:47:6 mol/mol)) and pH-sensitive liposomes (DOPE:CHEMS:CHOL: mPEG750-DSPE (43:21:30:6 mol/mol)) were compared in terms of size, charge, stability, pH-sensitivity and toxicity by inhibition of cell proliferation. Finally, confocal microscopy was used to study the cellular uptake of liposomes by three cell lines (Hs578t, WI-26 and MDA-MB-231), using 25-nitrobenzoxydiazol-cholesterol as a fluorescent marker of the vesicular membrane and rhodamine in the inner cavity of liposomes. Results: Sizes of 162.8 ± 4.6 nm and zeta potential of -9.3 ± 1.2 mV were obtained for standard liposomes (n=3) while the obtained values for pH-sensitive liposomes (n=3) were respectively of 184.8 ± 3.2 nm and -19.5 ± 2.6 mV. The two formulations were comparable in terms of shape and stability. Concerning the pH-sensitivity study, a significantly higher leakage of the encapsulated material was observed at pH 5 for pH-sensitive liposomes. Confocal pictures obtained with these vesicles on the three cell lines allowed us to visualize the colocalized red and green color with a higher concentration near the nucleus. Conclusion: Long circulating pH-sensitive liposomes are promising drug delivery systems in terms of cellular uptake. Experiments will be performed with biotinylated Print3G to assess its cellular distribution. Moreover, the accumulation of this formulation in breast tumor will be evaluated by in vivo studies