References of "Evrard, Brigitte"
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See detailNuclear delivery of a therapeutic peptide by long circulating pH-sensitive liposomes: Benefits over classical vesicles.
Ducat, Emilie ULg; Deprez, Julie ULg; Gillet, Aline ULg et al

in International Journal of Pharmaceutics (2011)

The purpose of this study is to propose a suitable vector combining increased circulation lifetime and intracellular delivery capacities for a therapeutic peptide. Long circulating classical liposomes ... [more ▼]

The purpose of this study is to propose a suitable vector combining increased circulation lifetime and intracellular delivery capacities for a therapeutic peptide. Long circulating classical liposomes [SPC:CHOL:PEG-750-DSPE (47:47:6 molar% ratio)] or pH-sensitive stealth liposomes [DOPE:CHEMS:CHOL:PEG(750)-DSPE (43:21:30:6 molar% ratio)] were used to deliver a therapeutic peptide to its nuclear site of action. The benefit of using stealth pH-sensitive liposomes was investigated and formulations were compared to classical liposomes in terms of size, shape, charge, encapsulation efficiency, stability and, most importantly, in terms of cellular uptake. Confocal microscopy and flow cytometry were used to evaluate the intracellular fate of liposomes themselves and of their hydrophilic encapsulated material. Cellular uptake of peptide-loaded liposomes was also investigated in three cell lines: Hs578t human epithelial cells from breast carcinoma, MDA-MB-231 human breast carcinoma cells and WI-26 human diploid lung fibroblast cells. The difference between formulations in terms of peptide delivery from the endosome to the cytoplasm and even to the nucleus was investigated as a function of time. Characterization studies showed that both formulations possess acceptable size, shape and encapsulation efficiency but cellular uptake studies showed the important benefit of the pH-sensitive formulation over the classical one, in spite of liposome PEGylation. Indeed, stealth pH-sensitive liposomes were able to deliver hydrophilic materials strongly to the cytoplasm. Most importantly, when encapsulated in pH-sensitive stealth liposomes, the peptide was able to reach the nucleus of tumorigenic and non tumorigenic breast cancer cells. [less ▲]

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See detailSkin penetration behaviour of liposomes as a function of their composition
Gillet, Aline ULg; Lecomte, Frédéric ULg; Hubert, Pascale ULg et al

in European Journal of Pharmaceutics & Biopharmaceutics (2011), 79

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See detailNear infrared and Raman spectroscopy as Process Analytical Technology tools for the manufacturing of silicone-based drug reservoirs
Mantanus, Jérôme ULg; Rozet, Eric ULg; Van Butsele, K. et al

in Analytica Chimica Acta (2011), 699

Using Near Infrared (NIR) and Raman spectroscopy as PAT tools, 3 critical quality attributes of a silicone-based drug reservoir were studied. First, the Active Pharmaceutical Ingredient (API) homogeneity ... [more ▼]

Using Near Infrared (NIR) and Raman spectroscopy as PAT tools, 3 critical quality attributes of a silicone-based drug reservoir were studied. First, the Active Pharmaceutical Ingredient (API) homogeneity in the reservoir was evaluated using Raman spectroscopy (mapping): the API distribution within the industrial drug reservoirs was found to be homogeneous while API aggregates were detected in laboratory scale samples manufactured with a non optimal mixing process. Second, the crosslinking process of the reservoirs was monitored at different temperatures with NIR spectroscopy. Conformity tests and Principal Component Analysis (PCA) were performed on the collected data to find out the relation between the temperature and the time necessary to reach the crosslinking endpoints. An agreement was found between the conformity test results and the PCA results. Compared to the conformity test method, PCA had the advantage to discriminate the heating effect from the crosslinking effect occurring together during the monitored process. Therefore the 2 approaches were found to be complementary. Third, based on the HPLC reference method, a NIR model able to quantify the API in the drug reservoir was developed and thoroughly validated. Partial Least Squares (PLS) regression on the calibration set was performed to build prediction models of which the ability to quantify accurately was tested with the external validation set. The 1.2 % RMSEP of the NIR model indicated the global accuracy of the model. The accuracy profile based on tolerance intervals was used to generate a complete validation report. The 95 % tolerance interval calculated on the validation results indicated that each future result will have a relative error below ±5 % with a probability of at least 95 %. In conclusion, 3 critical quality attributes of silicone-based drug reservoirs were quickly and efficiently evaluated by NIR and Raman spectroscopy. [less ▲]

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See detailOptimisation and validation of a fast HPLC method for the quantification of sulindac and its related impurities
Krier, Fabrice ULg; Brion, Michaël; Debrus, Benjamin ULg et al

in Journal of Pharmaceutical & Biomedical Analysis (2011), 54

The European Pharmacopoeia describes a liquid chromatography (LC) method for the quantification of sulindac, using a quaternary mobile phase including chloroform and with a rather long run time. In the ... [more ▼]

The European Pharmacopoeia describes a liquid chromatography (LC) method for the quantification of sulindac, using a quaternary mobile phase including chloroform and with a rather long run time. In the present study, a new method using a short sub-2μm column, which can be used on a classical HPLC system, was developed. The new LC conditions (without chloroform) were optimised by means of a new methodology based on design of experiments in order to obtain an optimal separation. Four factors were studied: the duration of the initial isocratic step, the percentage of organic modifier at the beginning of the gradient, the percentage of organic modifier at the end of the gradient and the gradient time. The optimal condition allows the separation of sulindac and of its 3 related impurities in six minutes instead of 18 min. Finally, the method was successfully validated using an accuracy profile approach in order to demonstrate its ability to accurately quantify these compounds. [less ▲]

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See detailCellular uptake of long-circulating pH-sensitive liposomes: evaluation of the liposome and its encapsulated material penetration in cancer cells
Ducat, Emilie ULg; Deprez, Julie ULg; Peulen, Olivier ULg et al

in Drug Discovery Today (2010, December), 15(23-24), 1079-1114

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 ... [more ▼]

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. [less ▲]

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See detailPEPTIDE-LOADED LIPOSOMES AGAINST BREAST CANCER: EFFECTIVE PENETRATION IN CELLS OF LONG CIRCULATING pH-SENSITIVE VESICLES
Ducat, Emilie ULg; Deprez, Julie ULg; Peulen, Olivier ULg et al

Poster (2010, October)

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 ... [more ▼]

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. [less ▲]

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See detailInterest of vesicular systems in dermal drug delivery
Gillet, Aline ULg; Lecomte, Frédéric ULg; Hubert, Pascale ULg et al

Poster (2010, October)

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See detailNIR AND RAMAN SPECTROSCOPY AS PAT TOOLS FOR THE MANUFACTURING OF SILICONE-BASED DRUG RESERVOIRS
Mantanus, Jérôme ULg; Ziemons, Eric ULg; Van Butsele, Kathy et al

Conference (2010, September 24)

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See detailOptimisation and validation of a fast HPLC method for the quantitation of sulindac and its impurities
Krier, Fabrice ULg; Brion, Michaël; Debrus, Benjamin ULg et al

Poster (2010, September)

Detailed reference viewed: 127 (43 ULg)
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See detailThe experimental design as practical approach to develop and optimize a formulation of peptide-loaded liposomes
Ducat, Emilie ULg; Brion, Michael; Lecomte, Frédéric ULg et al

in AAPS PharmSciTech (2010), 11(2), 966-975

To investigate the encapsulation of Print 3G, a peptidic agent that could reduce the angiogenic development of breast tumors, pegylated liposomes used as intravenous vectors were studied and characterized ... [more ▼]

To investigate the encapsulation of Print 3G, a peptidic agent that could reduce the angiogenic development of breast tumors, pegylated liposomes used as intravenous vectors were studied and characterized. Recently, the path of liposomes has been explored with success to improve the pharmacological properties of peptidic drugs and to stabilize them. In this study, loaded unilamellar vesicles composed of SPC:CHOL:mPEG2000-DSPE (47:47:6) were prepared by the hydration of lipid film technique. An HPLC method was developed and validated for the determination of Print 3G to calculate its encapsulation efficiency. Observed Print 3G adsorption on different materials employed during liposome preparation (such as glass beads, tubing, and connections for extrusion) led to the modification of the manufacturing method. The freeze-thawing technique was used to enhance the amount of Print 3G encapsulated into blank liposomes prepared using the hydration of lipid film procedure. Many factors may influence peptide entrapment, namely the number of freeze-thawing cycles, the lipid concentration, the peptide concentration, and the mixing time. Consequently, a design of experiments was performed to obtain the best encapsulation efficiency while minimizing the number of experiments. The lipid concentration and the number of freeze-thawing cycles were identified as the positive factors influencing the encapsulation. As a result of the optimization, an optimum was found and encapsulation efficiencies were improved from around 30% to 63%. Liposome integrity was evaluated by photon correlation spectroscopy and freeze-fracture electron microscopy to ensure that the selected formulation possesses the required properties to be a potential candidate for further in vitro and in vivo experiments. [less ▲]

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