Methylated N-(4-N,N-dimethylaminocinnamyl) chitosan enhances paracellular permeability across Caco-2 cells

Figures & data

Scheme 1.  Synthesis of N-(4-N,N-dimethylaminocinnamyl) chitosan.

Scheme 2.  Synthesis of methylated N-(4-N,N-dimethylaminocinnamyl) chitosan (TM-CM-CS).

Table 1.  Methylation of chitosan and N-(4-N,N-dimethylaminocinnamyl) chitosan (mean ± SD, n = 3).

Samples	ES (%)	DQT (%)		N(CH3)2 (%)	NHCH3(%)	TotalO-CH3 (%)	Recovery(%)
		DQAr(%)	DQCS(%)
TM64CS	—	—	64 ± 2	24 ± 2	5 ± 2	35 ± 2	74
4a	50 ± 2	50 ± 2	15 ± 2	24 ± 2	—	15 ± 2	90
4b	76 ± 2	76 ± 2	6 ± 2	10 ± 2	—	26 ± 2	109

ES is the extent of N-substitution; DQ_Ar is the degree of quaternization at aromatic substituents; DQ_CS is the degree of quaternization at the primary amino groups of CS; N(CH₃)₂ is N,N-dimethylation; NHCH₃ is N-methylation; Total O-CH₃ is the total degree of O-methylation of 3-O and 6-O at 3-hydroxyl and 6-hydroxyl positions of GlcN of chitosan, respectively; Recovery (%) is weight of product (g)/weight of starting reactant (g) × 100.

Figure 1.  ¹H NMR spectra of (1) chitosan, (3a) N-(4-N,N-dimethylaminocinnamyl) chitosan, and (4a) methylated N-(4-N,N-dimethylaminocinnamyl) chitosan with ES 50%.

Figure 2.  FTIR spectra of (1) chitosan, (3b) N-(4-N,N-dimethylaminocinnamyl) chitosan, (4b) methylated N-(4-N,N-dimethylaminocinnamyl) chitosan with ES 76% and TM-CS.

Figure 3.  The effect of TM-CM-CS on the TEER of Caco-2 cell monolayers: (a) TM₆₅CM₅₀CS, (b) TM₈₂CM₇₆CS. The TEER recovery pattern of the Caco-2 cell monolayers: (c) TM₆₅CM₅₀CS, (d) TM₈₂CM₇₆CS concentrations ranging from 0.05–5 mM, at pH 7.4 (♦) control, (▪) 0.05 mM, (▴) 0.5 mM, (◊) 1.25 mM, (□) 2.5 mM, (Δ) 5 mM. Each point represents the mean of three experiments. * p < 0.05.

Table 2.  The effect of the degree of quaternization (DQ), the extent of N-substitution (ES) of TM-CM-CS, the polymer concentration and the pH on TEER.

CS derivatives	pH	TEER (% of initial value) 2 h CS derivatives concentration (mM)
		0.05	0.5	1.25	2.5	5
TM65CM50CS (4a)	7.4	64.58 ± 1.5	23.56 ± 1.1	11.35 ± 0.2	9.57 ± 1.1	9.33 ± 0.8
TM82CM76CS (4b)	7.4	52.48 ± 0.8	25.22 ± 0.2	15.51 ± 0.5	13.81 ± 1.3	14.23 ± 0.4
TM64CS	7.4	73.06 ± 2.1	47.11 ± 1.1	13.44 ± 0.8	12.69 ± 0.6	11.23 ± 0.4
CSA	7.4	105.39 ± 3.1	104.63 ± 1.5	105.44 ± 2.1	107.36 ± 3.9	106.23 ± 2.8
CSA	6.2	81.35 ± 5.9	32.18 ± 4.8	21.07 ± 2.1	21.81 ± 2.5	16.58 ± 0.8

Figure 4.  The percentage of the reduction in TEER after 2 h incubation with 0.5 mM chitosan acetate (CSA) at pH 6.2 and 7.4 and chitosan derivatives at pH 7.4. Each point represents the mean of three experiments. * p < 0.05.

Table 3.  The effect of different degrees of quaternization (DQ) and the extent of N-substitution (ES) of TM-CM-CS on the transport of FD-4.

Samples	Cumulative transport of FD-4 at 4 h (µg)	Papp(10^−6cm/s)	R
Control	2.53 ± 0.1	0.16	1.00
TM64CS	3.40 ± 1.2	0.19	1.22
TM65CM50CS (4a)	12.71 ± 1.6	0.79	4.98
TM82CM76CS (4b)	8.11 ± 2.2	0.50	3.14

P_app is the apparent permeability coefficient; R is the absorption enhancement ratio.

Figure 5.  Cumulative transport of FD-4 in the presence of 0.5 mM TM-CM-CS at pH 7.4 for 4 h: (♦) control, (x) TM₆₄CS, (▴) TM₆₅CM₅₀CS, (▪) TM₈₂CM₇₆CS. Each point represents the mean of three experiments. * p < 0.05.

Table 4.  Cytotoxicity TM-CM-CS incubated with Caco-2 cells for 2 and 24 h (n = 8).

Samples	pH	IC50 (mM) at 2 h	IC50 (mM) at 24 h
TM65CM50CS (4a)	7.4	> 5	0.14 ± 0.01
TM82CM76CS (4b)	7.4	> 5	0.12 ± 0.01
TM64CS	7.4	3.30 ± 1.02	0.14 ± 0.01
CSA	6.2	> 5	0.90 ± 0.01
CSA	7.4	> 5	> 5