Applications of cyclodextrins for skin formulation and delivery

in Bilensoy, Erem (Ed.) Cyclodextrins in pharmaceutics, cosmetics and biomedicine (2011)

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

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 ▲]

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

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 ▲]

POLYPLEXES TARGETING E6: OPTIMIZATION OF THE FORMULATIONS IN TERMS OF TRANSFECTION, TOXICITY AND EFFICIENCY

Poster (2010, October)

ESR technique for non invasive way to quantify cyclodextrins effect on cell membranes

in Biochemical and Biophysical Research Communications (2010)

A new way to study the action of cyclodextrin was developed to quantify the damage caused on cell membrane and lipid bilayer. The Electron Spin Resonance (ESR) spectroscopy was used to study the action of ... [more ▼]

A new way to study the action of cyclodextrin was developed to quantify the damage caused on cell membrane and lipid bilayer. The Electron Spin Resonance (ESR) spectroscopy was used to study the action of Randomly methylated-beta-cyclodextrin (Rameb) on living cells (HCT-116). The relative anisotropy observed in ESR spectrum of nitroxide spin probe (5-DSA and cholestane) is directly related to the rotational mobility of the probe,which can be further correlated with themicroviscosity. The use of ESR probes clearly shows a close correlation between cholesterol contained in cells and cellular membrane microviscosity. This study also demonstrates the Rameb ability to extract cholesterol and phospholipids in time- and dose-dependent ways. In addition, ESR spectra enabled to establish that cholesterol is extracted from lipid rafts to form stable aggregates. The present work supports that ESR is an easy, reproducible and noninvasive technique to study the effect of cyclodextrins on cell membranes. [less ▲]

IESR spectroscopy: an easy and non invasive way to determine the extraction power of beta-Cyclodextrins on membranes

Conference (2010, May 10)

Classical and deformable liposomes: possible mechanism of penetration through pig ear skin

Poster (2010, March 29)

Comparison of chitosan/siRNA and trimethylchitosan/siRNA complexes behaviour in vitro

in International Journal of Biological Macromolecules (2010), 46

Chitosan and trimethylchitosan (TMC)-siRNA nanoparticles were produced by simple complexation technique or by ionic gelation using tripolyphosphate (TPP). The obtained complexes were characterized in ... [more ▼]

Chitosan and trimethylchitosan (TMC)-siRNA nanoparticles were produced by simple complexation technique or by ionic gelation using tripolyphosphate (TPP). The obtained complexes were characterized in terms of physicochemical properties such as size, zeta potential, complexation efficiency and stability. Furthermore, cytotoxicity, cell uptake and transfection efficiency of polyplexes were evaluated in vitro. Under pH condition of cell culture medium, a strong decrease in siRNA condensation efficiency was observed with chitosan nanoparticles. This characteristic resulted in low transfection efficiencies in HEK293 cell line. Formulation of chitosan polyplexes with TPP led to improvement of polyplexes stability but no significant increase in transfection efficiency was observed compared to simple chitosan complexes. By contrast, TMC complexes did not have pH dependency on siRNA complexation. TMCsiRNA nanoparticles were stable in physiological condition. Accordingly, cellular uptake was increased compared to chitosan polyplexes. However, improvement of transfection efficiency was low regarding to cellular uptake of these complexes. Chitosan and TMC complexes present some characteristics favourable for siRNA delivery, such as ability to integrate siRNA into small discrete particles or low toxicity of the complexes. This study also highlights the importance of complexes stability in physiological environment for siRNA transfection purposes. [less ▲]

Development and validation of a sensitive solid phase extraction/hydrophilic interaction liquid chromatography/mass spectrometry method for the accurate determination of glucosamine in dog plasma.

in Journal of Chromatography. A (2010), 1217

A sensitive and accurate LC/MS method was developed for the monitoring of glucosamine (GLcN) dog plasmatic concentration. In this scope, relatively low plasmatic concentrations of GLcN were expected ... [more ▼]

A sensitive and accurate LC/MS method was developed for the monitoring of glucosamine (GLcN) dog plasmatic concentration. In this scope, relatively low plasmatic concentrations of GLcN were expected, ranging from 50 to 1000ng/mL. Liquid chromatography coupled to simple quadrupole mass spectrometry detection (LC/MS) was selected bringing the selectivity and the sensitivity needed for this application. Additionally, a solid phase extraction (SPE) step was performed to reduce matrix and ion suppression effects. Due to the ionisable character of the compound of interest, a mixed-mode strong cation exchange (Plexa PCX) disposable extraction cartridge (DEC) was selected. The separation was carried out on a Zorbax SB-CN column (5mum, 4.6mm i.d.x250mm), considering hydrophilic interaction liquid chromatography (HILIC). Indeed, the mobile phase was made of methanol and 5mM ammonium hydrogen carbonate buffer at pH 7.5 (95/5, v/v). The detection was led at m/z ratios of 180.0 and 417.0, for GLcN and IS, respectively. Reliability of the results was demonstrated through the validation of the method using an approach based on the accuracy profile allowing managing the risk associated to the use of these methods in routine analysis: it is thus guaranteed that each future result will fall in the +/-30% acceptance limits with a probability of at least 90%. Successful application of the method to a preliminary pharmacokinetic study illustrated the usefulness of the method for pre-clinical studies. [less ▲]

Long-circulating liposomes: comparison between pH-sensitive and standard vesicles in terms of cellular uptake

Conference (2010)