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Chemical coupling of thiolated chitosan to preformed liposomes improves mucoadhesive properties.

Gradauer K, Vonach C, Leitinger G, Kolb D, Fröhlich E, Roblegg E, Bernkop-Schnürch A, Prassl R - Int J Nanomedicine (2012)

Bottom Line: Likewise, their zeta potentials gradually increased from about -38 mV to +20 mV, clearly indicating an effective coupling of chitosan-TGA to the surface of liposomes.As a result of mucoadhesion tests, we found an almost two-fold increase in the mucoadhesion of coupled liposomes relative to uncoupled ones.Taken together, our current results indicate that thiomer-coated liposomes possess a high potential to be used as an oral drug-delivery system.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biophysics and Nanosystems Research, Austrian Academy of Sciences, Graz, Austria.

ABSTRACT

Aim: To develop mucoadhesive liposomes by anchoring the polymer chitosan-thioglycolic acid (chitosan-TGA) to the liposomal surface to target intestinal mucosal membranes.

Methods: Liposomes consisting of phosphatidylcholine (POPC) and a maleimide-functionalized lipid were incubated with chitosan-TGA, leading to the formation of a thioether bond between free SH-groups of the polymer and maleimide groups of the liposome. Uncoated and newly generated thiomer-coated liposomes were characterized according to their size, zeta potential, and morphology using photon correlation spectroscopy and transmission electron microscopy. The release behavior of calcitonin and the fluorophore/quencher-couple ANTS/DPX (8-aminonaphthalene-1,3,6-trisulfonic acid/p-xylene-bis- pyridinium bromide) from coated and uncoated liposomes, was investigated over 24 hours in simulated gastric and intestinal fluids. To test the mucoadhesive properties of thiomer-coated and uncoated liposomes in-vitro, we used freshly excised porcine small intestine.

Results: Liposomes showed a concentration-dependent increase in size - from approximately 167 nm for uncoated liposomes to 439 nm for the highest thiomer concentration used in this study. Likewise, their zeta potentials gradually increased from about -38 mV to +20 mV, clearly indicating an effective coupling of chitosan-TGA to the surface of liposomes. As a result of mucoadhesion tests, we found an almost two-fold increase in the mucoadhesion of coupled liposomes relative to uncoupled ones. With fluorescence microscopy, we saw a tight adherence of coated particles to the intestinal mucus.

Conclusion: Taken together, our current results indicate that thiomer-coated liposomes possess a high potential to be used as an oral drug-delivery system.

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Related in: MedlinePlus

Transmission electron micrographs using freeze fracturing of (A) uncoated POPC/DOPE-MCC liposomes and (B) POPC/DOPE-MCC liposomes coated with chitosan-TGA (4:1 molar ratio of SH-groups to maleimide groups).Notes: Arrows indicate the polymer coat. Magnification: 30,000×. Scale bar indicates 200 nm.Abbreviations: DOPE-MCC, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide]; POPC, Palmitoyl-oleoyl-phosphatidylcholine; TGA, thioglycolic acid.
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f6-ijn-7-2523: Transmission electron micrographs using freeze fracturing of (A) uncoated POPC/DOPE-MCC liposomes and (B) POPC/DOPE-MCC liposomes coated with chitosan-TGA (4:1 molar ratio of SH-groups to maleimide groups).Notes: Arrows indicate the polymer coat. Magnification: 30,000×. Scale bar indicates 200 nm.Abbreviations: DOPE-MCC, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide]; POPC, Palmitoyl-oleoyl-phosphatidylcholine; TGA, thioglycolic acid.

Mentions: To study the morphology of coated and uncoated liposomes, negative staining transmission electron microscopy was chosen. Ammonium molybdate (5%), phosphotungstic acid (1%), and uranyl acetate (2%) were tried as staining agents. Uranyl acetate was found to give the best contrast and the most homogeneous distribution of the dye and was used for all further preparations. Pictures of uncoated liposomes showed mostly spherical liposomes (170–200 nm mean size). After adding the polymer, the liposomes were apparently linked with each other (Figure 5). With this technique, we were able to rule out the possibility that the increase in size of the liposomes, after adding the polymer, was caused by fusion. Nevertheless, we were not able to detect the polymer coat itself in negative-contrasted samples. Therefore, freeze fracturing was used to reveal the polymer coat (Figure 6). Drying and staining steps, during which the liposomes may suffer from different pH or salt conditions, are not necessary; however, images prepared by freeze fracturing do not reflect the real size of the liposomes, since fracturing does not have to occur through the center of the particles.36 The freeze fractures of the coated samples clearly revealed the presence of the polymer at the periphery of the liposomes, which is marked by arrows in Figure 6B. Although no association of liposomes was found in uncoated samples (Figure 6A), single liposomes – as well as aggregates, which involve about three to ten liposomes – were visible in the coated sample (Figure 6B). Henriksen et al observed equally heterogeneous aggregates after adding negatively charged liposomes to a chitosan solution using cryo-electron microscopy.35 This heterogeneity is also reflected in the polydispersity index of the size measurements, which increased with increasing the amount of polymer.


Chemical coupling of thiolated chitosan to preformed liposomes improves mucoadhesive properties.

Gradauer K, Vonach C, Leitinger G, Kolb D, Fröhlich E, Roblegg E, Bernkop-Schnürch A, Prassl R - Int J Nanomedicine (2012)

Transmission electron micrographs using freeze fracturing of (A) uncoated POPC/DOPE-MCC liposomes and (B) POPC/DOPE-MCC liposomes coated with chitosan-TGA (4:1 molar ratio of SH-groups to maleimide groups).Notes: Arrows indicate the polymer coat. Magnification: 30,000×. Scale bar indicates 200 nm.Abbreviations: DOPE-MCC, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide]; POPC, Palmitoyl-oleoyl-phosphatidylcholine; TGA, thioglycolic acid.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3367495&req=5

f6-ijn-7-2523: Transmission electron micrographs using freeze fracturing of (A) uncoated POPC/DOPE-MCC liposomes and (B) POPC/DOPE-MCC liposomes coated with chitosan-TGA (4:1 molar ratio of SH-groups to maleimide groups).Notes: Arrows indicate the polymer coat. Magnification: 30,000×. Scale bar indicates 200 nm.Abbreviations: DOPE-MCC, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide]; POPC, Palmitoyl-oleoyl-phosphatidylcholine; TGA, thioglycolic acid.
Mentions: To study the morphology of coated and uncoated liposomes, negative staining transmission electron microscopy was chosen. Ammonium molybdate (5%), phosphotungstic acid (1%), and uranyl acetate (2%) were tried as staining agents. Uranyl acetate was found to give the best contrast and the most homogeneous distribution of the dye and was used for all further preparations. Pictures of uncoated liposomes showed mostly spherical liposomes (170–200 nm mean size). After adding the polymer, the liposomes were apparently linked with each other (Figure 5). With this technique, we were able to rule out the possibility that the increase in size of the liposomes, after adding the polymer, was caused by fusion. Nevertheless, we were not able to detect the polymer coat itself in negative-contrasted samples. Therefore, freeze fracturing was used to reveal the polymer coat (Figure 6). Drying and staining steps, during which the liposomes may suffer from different pH or salt conditions, are not necessary; however, images prepared by freeze fracturing do not reflect the real size of the liposomes, since fracturing does not have to occur through the center of the particles.36 The freeze fractures of the coated samples clearly revealed the presence of the polymer at the periphery of the liposomes, which is marked by arrows in Figure 6B. Although no association of liposomes was found in uncoated samples (Figure 6A), single liposomes – as well as aggregates, which involve about three to ten liposomes – were visible in the coated sample (Figure 6B). Henriksen et al observed equally heterogeneous aggregates after adding negatively charged liposomes to a chitosan solution using cryo-electron microscopy.35 This heterogeneity is also reflected in the polydispersity index of the size measurements, which increased with increasing the amount of polymer.

Bottom Line: Likewise, their zeta potentials gradually increased from about -38 mV to +20 mV, clearly indicating an effective coupling of chitosan-TGA to the surface of liposomes.As a result of mucoadhesion tests, we found an almost two-fold increase in the mucoadhesion of coupled liposomes relative to uncoupled ones.Taken together, our current results indicate that thiomer-coated liposomes possess a high potential to be used as an oral drug-delivery system.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biophysics and Nanosystems Research, Austrian Academy of Sciences, Graz, Austria.

ABSTRACT

Aim: To develop mucoadhesive liposomes by anchoring the polymer chitosan-thioglycolic acid (chitosan-TGA) to the liposomal surface to target intestinal mucosal membranes.

Methods: Liposomes consisting of phosphatidylcholine (POPC) and a maleimide-functionalized lipid were incubated with chitosan-TGA, leading to the formation of a thioether bond between free SH-groups of the polymer and maleimide groups of the liposome. Uncoated and newly generated thiomer-coated liposomes were characterized according to their size, zeta potential, and morphology using photon correlation spectroscopy and transmission electron microscopy. The release behavior of calcitonin and the fluorophore/quencher-couple ANTS/DPX (8-aminonaphthalene-1,3,6-trisulfonic acid/p-xylene-bis- pyridinium bromide) from coated and uncoated liposomes, was investigated over 24 hours in simulated gastric and intestinal fluids. To test the mucoadhesive properties of thiomer-coated and uncoated liposomes in-vitro, we used freshly excised porcine small intestine.

Results: Liposomes showed a concentration-dependent increase in size - from approximately 167 nm for uncoated liposomes to 439 nm for the highest thiomer concentration used in this study. Likewise, their zeta potentials gradually increased from about -38 mV to +20 mV, clearly indicating an effective coupling of chitosan-TGA to the surface of liposomes. As a result of mucoadhesion tests, we found an almost two-fold increase in the mucoadhesion of coupled liposomes relative to uncoupled ones. With fluorescence microscopy, we saw a tight adherence of coated particles to the intestinal mucus.

Conclusion: Taken together, our current results indicate that thiomer-coated liposomes possess a high potential to be used as an oral drug-delivery system.

Show MeSH
Related in: MedlinePlus