Vitamin E succinate-grafted-chitosan/chitosan oligosaccharide mixed micelles loaded with C-DMSA for Hg²⁺ detection and detoxification in rat liver

Figures & data

Figure 1 Structure of DMSA (A) and design of fluorescent theranostic agents for Hg²⁺(B).

Abbreviations: DMSA, meso-2,3-dimercaptosuccinic acid; C-DMSA, 3-formyl-7-diethylamino coumarin masked meso-dimercaptosuccinic acid.

Figure 2 Synthesis of VES-g-CS (A), ¹H-NMR (B) and FT-IR (C) of VES-g-CS.

Abbreviations: VES, vitamin E succinate; CS, chitosan; VES-g-CS, vitamin E succinate-grafted-chitosan; FT-IR, Fourier transform infrared; HOBt, 1-Hydroxybenzotriazole.

Figure 3 Transmission electron microscopy of C-DMSA@VES-g-CSO/VES-g-CS MM (A); particle size distribution of C-DMSA@VES-g-CSO/VES-g-CS MM obtained from dynamic light scattering (B); C-DMSA release profile from micelles in PBS solution (pH=7.4) (C) and particle size changes of C-DMSA loaded micelles stored at 4°C as a function of storage time (D).

Note: Data were presented as mean±SD (n=3).

Abbreviations: C-DMSA, 3-formyl-7-diethylamino coumarin masked meso-dimercaptosuccinic acid; C-DMSA@VES-g-CSO micelles, C-DMSA loaded vitamin E succinate-grafted-chitosan oligosaccharide micelles; C-DMSA@VES-g-CS micelles, C-DMSA loaded vitamin E succinate-grafted-chitosan micelles; C-DMSA@VES-g-CSO/VES-g-CS MM, C-DMSA loaded vitamin E succinate-grafted-chitosan oligosaccharide/vitamin E succinate-grafted-chitosan mixed micelles.

Figure 4 Fluorescence microscopy images (A) and flow cytometry (B) of L929 cells after incubation with PBS.

Notes: Blue, cell nuclei stained with Hoechst 33258; green, fluorescence of coumarin-aldehyde generated by C-DMSA after its reaction with Hg²⁺.

Abbreviations: C-DMSA, 3-formyl-7-diethylamino coumarin masked meso-dimercaptosuccinic acid; C-DMSA@VES-g-CSO/VES-g-CS MM, C-DMSA loaded vitamin E succinate-grafted-chitosan oligosaccharide/vitamin E succinate-grafted-chitosan mixed micelles.

Figure 5 Fluorescence imaging intracellular Hg²⁺ of L929 cells with free C-DMSA or C-DMSA loaded mixed micelles (A); cell viability of L929 cells treated with Hg²⁺ and different treatments with untreated cells and poisoning group served as controls (B).

Notes: Left column, cells without Hg²⁺ pretreatment; right column, cells with Hg²⁺ pretreatment; top row, cells treated with PBS (blank); middle row, cell treated with C-DMSA (20 μg/mL); bottom row, cells treated with C-DMSA@VES-g-CSO/VES-g-CS MM. **p<0.01, compared to control group; ^##p<0.01, compared to poisoning group. Data were presented as mean±SD (n=6).

Abbreviations: DMSA, meso-2,3-dimercaptosuccinic acid; C-DMSA; 3-formyl-7-diethylamino coumarin masked meso-dimercaptosuccinic acid; C-DMSA@VES-g-CSO/VES-g-CS MM, C-DMSA loaded micelles, vitamin E succinate-grafted-chitosan oligosaccharide/vitamin E succinate-grafted-chitosan mixed micelles.

Figure 6 Ex vivo fluorescent photograph of major organs dissected from IR-775@VES-g-CSO/VES-g-CS MM treated mice (A). Ex vivo fluorescent photograph of major organs dissected from mice with different treatments (B).

Notes: Blank control (a); HgCl₂ poisoned (b); 15 mg/kg of C-DMSA@VES-g-CSO/VES-g-CS MM (c); HgCl₂ poisoned with C-DMSA treatment (d); HgCl₂ poisoned with 15 mg/kg of C-DMSA@VES-g-CSO/VES-g-CS MM treatment (e); HgCl₂ poisoned with 30 mg/kg of C-DMSA@VES-g-CSO/VES-g-CS MM treatment (f).

Abbreviations: IR-775 loaded micelles, vitamin E succinate-grafted-chitosan oligosaccharide/vitamin E succinate-grafted-chitosan mixed micelles.

Figure 7 ALT (A) and AST (B) in blood samples from rats with different treatments.

Notes: *p<0.05, compared to control group; ^#p<0.05, compared to HgCl₂ group. Data were presented as mean±SD (n=6).

Abbreviations: ALT, alanine transaminase; AST, aspartate transaminase; C-DMSA; 3-formyl-7-diethylamino coumarin masked meso-dimercaptosuccinic acid; C-DMSA loaded MM, C-DMSA loaded vitamin E succinate-grafted-chitosan oligosaccharide/vitamin E succinate-grafted-chitosan mixed micelles.

Figure S1 ¹H-NMR CS (A) and VES (B).

Abbreviations: VES, vitamin E succinate; CS, chitosan.

Figure S2 Determination of CMC values of VES-g-CSO/VES-g-CS (w/w=4:1) (A), VES-g-CSO (B) and VES-g-CS (C) solutions.

Abbreviations: CMC, critical micelle concentration; VES-g-CSO, vitamin E succinate-grafted-chitosan oligosaccharide; VES-g-CS, vitamin E succinate-grafted-chitosan; VES-g-CSO/VES-g-CS, vitamin E succinate-grafted-chitosan oligosaccharide/vitamin E succinate-grafted-chitosan.

Figure S3 Cell viability of L929 cells treated with the blank mixed micelles (VES-g-CSO/VES-g-CS MM) or C-DMSA@VES-g-CSO/VES-g-CS MM (A). Cell viability of L929 cells treated with C-DMSA or C-DMSA@VES-g-CSO/VES-g-CS MM (B).

Note: Data were presented as mean±SD (n=3).

Abbreviations: C-DMSA, 3-formyl-7-diethylamino coumarin masked meso-dimercaptosuccinic acid; VES-g-CSO/VES-g-CS MM, vitamin E succinate-grafted-chitosan oligosaccharide/vitamin E succinate-grafted-chitosan mixed micelles; C-DMSA@VES-g-CSO/VES-g-CS MM, C-DMSA loaded vitamin E succinate-grafted-chitosan oligosaccharide/vitamin E succinate-grafted-chitosan mixed micelles.

Figure S4 Mean plasma concentration-time curves of C-DMSA after intravenous administration of C-DMSA and C-DMSA@VES-g-CSO/VES-g-CS MM in rats.

Notes: All the rats were received the single dosage at an equivalent dose of 10 mg/kg C-DMSA. Data were presented as mean±SD (n=3).

Abbreviations: C-DMSA, 3-formyl-7-diethylamino coumarin masked meso-dimercaptosuccinic acid; C-DMSA@VES-g-CSO/VES-g-CS MM, C-DMSA loaded vitamin E succinate-grafted-chitosan oligosaccharide/vitamin E succinate-grafted-chitosan mixed micelles.

Table 1 Physicochemical properties of C-DMSA loaded micelles

Micelles	LE (%)	EE (%)	Particle size (nm)	Zeta potential (mV)	PI
VES-g-CSO micelles	/	/	178.5±2.1	35.7±3.2	0.28
C-DMSA@VES-g-CSO micelles	7.5±0.2	82.8±1.5	185.3±6.2	30.9±1.9	0.21
VES-g-CS micelles	/	/	116.9±1.1	17.3±1.7	0.17
C-DMSA@VES-g-CS micelles	3.1±0.2	34.9±1.2	120.9±3.1	17.3±0.4	0.17
VES-g-CSO/VES-g-CS MM	/	/	124.5±3.5	25.6±0.9	0.16
C-DMSA@VES-g-CSO/VES-g-CS MM	6.9±0.3	76.6±1.7	133.2±4.9	24.1±0.1	0.16

Notes: Data were presented as mean±SD (n=3). “/”means the data is indeterminable.

Abbreviations: LE, loading efficiency; EE, entrapment efficiency; PI, polydispersity index; VES-g-CSO micelles, vitamin E succinate-grafted-chitosan oligosaccharide micelles; VES-g-CS micelles, vitamin E succinate-grafted-chitosan micelles; VES-g-CSO/VES-g-CS MM, vitamin E succinate-grafted-chitosan oligosaccharide/vitamin E succinate-grafted-chitosan mixed micelles; C-DMSA, 3-formyl-7-diethylamino coumarin masked meso-dimercaptosuccinic acid.

Table 2 Liver coefficients and contents of total mercury in rat liver and blood

Treatment	Mercury		Liver coefficients (%)
	Liver (μg/g tissue)	Blood (μg/L)
Control	0.01±0.01	0±0.01	4.57±0.11
HgCl2	4.57±0.42***	0.86±0.18***	5.12±0.11*
HgCl2+C-DMSA (15 mg/kg)	3.64±0.54	0.70±0.19	4.93±0.15
HgCl2+C-DMSA loaded micelles (15 mg/kg)	2.63±0.55^#	0.59±0.06^#	4.64±0.19^#
HgCl2+C-DMSA loaded micelles (30 mg/kg)	2.38±0.29^#	0.52±0.02^#	4.59±0.13^#

Notes: *p<0.05, ***p<0.001, compared to control group. ^#p<0.05 compared to HgCl₂ group. Data were presented as mean±SD (n=6).

Abbreviations: C-DMSA, 3-formyl-7-diethylamino coumarin masked meso-dimercaptosuccinic acid; C-DMSA loaded micelles, C-DMSA loaded vitamin E succinate-grafted-chitosan oligosaccharide/vitamin E succinate-grafted-chitosan mixed micelles.

Table S1 Elemental analysis of VES-g-CSO and VES-g-CS

Polymer	N%	C%	H%	DS (%)
CSO	6.4±0.1	34.1±0.2	6.6±0.1	/
VES-g-CSO	4.5±0.2	34.5±0.2	6.8±0.1	1.2±0.3
CS	7.3±0.0	40.1±0.1	7.2±0.1	/
VES-g-CS	6.4±0.1	40.3±0.8	7.1±0.1	3.0±0.8

Notes: Data were presented as mean ± SD (n=3). “/”means the data is indeterminable.

Abbreviations: DS (%), degree of substitution of VES to amino group of CSO or CS was calculated by elemental analysis; CS, chitosan; CSO, chitosan oligosaccharide; VES-g-CSO, vitamin E succinate- grafted-chitosan oligosaccharide; VES-g-CS, vitamin E succinate-grafted-chitosan.

Table S2 Pharmacokinetic parameters of C-DMSA after a single dosage intravenous administration to rats

Pharmacokinetic parameters	C-DMSA	C-DMSA@VES-g-CSO/VES-g-CS MM
t1/2α (h)	0.12±0.01	0.74±0.12**
t1/2β (h)	1.91±0.20	15.23±1.20**
Vd (L/kg)	0.21±0.06	0.17±0.04
AUC0-24h (μg·h/L)	9.96±0.26	45.59±1.37***
CL (L/h/kg)	0.49±0.07	0.10±0.02**

Notes: All the rats were received the single dosage at an equivalent dose of C-DMSA (10 mg/kg). **p<0.01, ***p<0.001, compared to C-DMSA group. Data were presented as mean±SD (n=3).

Abbreviations: t_1/2α, a rapid distribution half-life; t_1/2β, elimination half-life; AUC, the area under the concentration-time curve; V_d, the apparent volume of the central chamber; CL, clearance; C-DMSA, 3-formyl-7-diethylamino coumarin masked meso-dimercaptosuccinic acid; C-DMSA@VES-g-CSO/VES-g-CS MM, C-DMSA loaded vitamin E succinate-grafted-chitosan oligosaccharide/vitamin E succinate-grafted-chitosan mixed micelles.