Chemical coupling of thiolated chitosan to preformed liposomes improves mucoadhesive properties

Figures & data

Figure 1 Reaction scheme for the covalent coupling of chitosan-TGA to a maleimide-functionalized phospholipid to form a stable thioether bond.

Abbreviations: DOPE-MCC, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide]; TGA, thioglycolic acid.

Figure 2 Falling Liquid Film technique to measure the mucoadhesion of coated and uncoated liposomes.

Figure 3 Particle size after the addition of different amounts of polymers (given by the molar ratio of SH-groups of the polymer to maleimide groups of the liposome) to POPC liposomes (○) and POPC/DOPE-MCC liposomes (■).

Note: Indicated values are means ± standard deviation of at least three measurements.

Abbreviations: DOPE-MCC, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N- [4-(p-maleimidomethyl)cyclohexane-carboxamide]; POPC, Palmitoyl-oleoylphosphatidylcholine; TC, chitosan-thioglycolic acid.

Figure 4 Zeta potential after the addition of different amounts of polymers (given by the molar ratio of SH-groups of the polymer to maleimide groups of the liposome) to POPC liposomes (○) and POPC/DOPE-MCC liposomes (■).

Note: Indicated values are means ± standard deviation of at least three measurements.

Abbreviations: DOPE-MCC, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine- N-[4-(p-maleimidomethyl)cyclohexane-carboxamide]; POPC, Palmitoyl-oleoylphosphatidylcholine; TC, chitosan-thioglycolic acid.

Table 1 Amount of SH-groups after coupling

	Coupled liposomes 4:1	Control liposomes 4:1	Coupled liposomes 6:1	Control liposomes 6:1
Amount of SH-groups (nmol/mg total lipid)	283 ± 40	382 ± 19	494 ± 29	594 ± 12

Notes: Coupled liposomes (POPC/DOPE-MCC) with a coupling ratio (SH-groups to maleimide groups) of 4:1 and 6:1, and control liposomes (POPC) with the same amount of polymer solution added. All values are means ± standard deviation of five experiments.

Abbreviations: DOPE-MCC, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide]; POPC, Palmitoyl-oleoylphosphatidylcholine.

Figure 5 Transmission electron micrographs with negative staining technique 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: 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.

Figure 6 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.

Figure 7 Release of ANTS/DPX at predetermined time points from uncoated POPC/DOPE-MCC liposomes (○) and coated POPC/DOPE-MCC liposomes (4:1 molar ratio of SH-groups to maleimide groups) (■) in simulated gastric fluid (A) and simulated intestinal fluid (B).

Note: Each point represents the mean value of two different determinations.

Abbreviations: ANTS, anionic fluorophore 8-aminonaphthalene-1,3,6-trisulfonic acid; DOPE-MCC, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl) cyclohexane-carboxamide]; DPX, p-xylene-bis-pyridinium bromide; POPC, Palmitoyl-oleoyl-phosphatidylcholine.

Figure 8 Release of FITC-calcitonin at predetermined time points from uncoated POPC/DOPE-MCC liposomes (○) and coated POPC/DOPE-MCC liposomes (4:1 molar ratio of SH-groups to maleimide groups) (■) in simulated gastric fluid (A) and simulated intestinal fluid (B).

Note: Results are means ± standard deviation (n = 3).

Abbreviations: DOPE-MCC, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide]; POPC, Palmitoyl-oleoyl-phosphatidylcholine.

Table 2 Percentage of uncoated/coated liposomes bound to the tissue after using the falling liquid film technique

Mucoadhesion of coated liposomes (%)	Mucoadhesion of uncoated liposomes (%)	Improvement ratio
(A) 25 μg/mL
70.4	33.5	2.1
68.4	46.1	1.5
61.3	41.7	1.5
(B) 100 μg/mL
56.6	31.4	1.8
60.3	45.0	1.3
60.0	26.0	2.3

Notes: Comparison of three independent tests; 1 mL of coated or uncoated liposomes with a concentration of (A) 25 μg/mL or (B) 100 μg/mL was applied to the tissue and the amount of bound particles was calculated as a percentage (%) of the total.

Figure 9 Histological sections of porcine small intestine. (A) untreated intestinal tissue (negative control), (B) tissue treated with POPC/DOPE-Liss Rhod liposomes and (C) with POPC/DOPE-Liss Rhod/DOPE-MCC liposomes coated with chitosan-TGA (molar ratio of SH-groups to maleimide-groups: 4:1).

Abbreviations: DOPE-Liss Rhod, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl); DOPE-MCC, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide]; POPC, Palmitoyl-oleoyl-phosphatidylcholine; TGA, thioglycolic acid.