Synthesis and characterization of low-toxicity N-caprinoyl-N-trimethyl chitosan as self-assembled micelles carriers for osthole

Figures & data

Figure 1 Synthesis route of TMC and CA-TMC.

Abbreviations: CA-TMC2, N-caprinoyl-N-trimethyl chitosan; TMC, N-trimethyl chitosan.

Figure 2 FTIR spectra of chitosan (A), TMC2 (B), and CA-TMC2 (C).

Abbreviations: CA-TMC2, N-caprinoyl-N-trimethyl chitosan; FTIR; Fourier transform infrared; TMC, N-trimethyl chitosan.

Figure 3 ¹H-NMR spectra of chitosan (A), TMC2 (B), and CA-TMC2 (C) in D₂O.

Abbreviations: CA-TMC, N-caprinoyl-N-trimethyl chitosan; ¹H-NMR, proton nuclear magnetic resonance; TMC, N-trimethyl chitosan.

Figure 4 ¹H-NMR spectra of capric acid (A) and CA-TMC2 (B) in DMSO-d6.

Abbreviations: CA-TMC, N-caprinoyl-N-trimethyl chitosan; DMSO, dimethyl sulfoxide; ¹H-NMR, proton nuclear magnetic resonance.

Table 1 Chemical compositions and properties of polymer micelles (n = 3)

Sample	N(C)/N(N)	Standard deviation (%)	Degree of quaternization (%)	Degree of substitution (%)
CS	5.5	0.2	–	–
TMC1	6.3	4.2	27.0	–
TMC2	6.8	2.2	43.1	–
TMC3	7.9	3.8	62.0	–
CA-TMC1	10.8	3.8	–	44.9
CA-TMC2	9.5	2.7	–	27.0
CA-TMC3	9.2	1.6	–	13.3

Notes: N(C)/N(N), the rate of the number of elemental carbon to the number of elemental nitrogen in polymer.

Abbreviations: CA-TMC, N-caprinoyl-N-trimethyl chitosan; CS, chitosan; TMC, N-trimethyl chitosan.

Figure 5 Coordinates of CA-TMC1 (A), CA-TMC2 (B), and CA-TMC3 (C) micellar solution at different concentrations.

Abbreviation: CA-TMC, N-caprinoyl-N-trimethyl chitosan.

Figure 6 Cytocompatibility of CA-TMC1 CA-TMC2 and CA-TMC3 at different concentrations on Hela, MCF-7, and HL7702 cells.

Notes: The cells were identified using micelles for 48 hours. Data are presented as mean ± SD (n = 5).

Abbreviations: CA-TMC, N-caprinoyl-N-trimethyl chitosan; SD, standard deviation.

Figure 7 Hemolysis evaluation of Tween 80, CA-TMC1, CA-TMC2, CA-TMC3, and F188 using erythrocytes freshly harvested from rabbit blood.

Abbreviation: CA-TMC, N-caprinoyl-N-trimethyl chitosan.

Figure 8 Tissue slice of the physiological saline group and the CA-TMC2 group (×100).

Abbreviation: CA-TMC2, N-caprinoyl-N-trimethyl chitosan.

Table 2 Characteristics of OST/CA-TMC micelles

Sample	CA-TMC micelles		OST/CA-TMC micelles
	Zeta potential (mV)	Size (nm)	Zeta potential (mV)	Size (nm)	Polydispersity index	EE (%)	DL (%)
CA-TMC1	35.1	212.7 ± 26.1	32.9	285.5 ± 32.9	0.3	73.2	17.8
CA-TMC2	48.6	168.3 ± 19.0	43.7	192.0 ± 25.4	0.2	79.1	19.1
CA-TMC3	56.8	136.5 ± 15.4	52.4	184.7 ± 20.9	0.2	64.3	16.1

Abbreviations: CA-TMC, N-caprinoyl-N-trimethyl chitosan; DL, drug loading; EE, encapsulation efficiency; OST/CA-TMC, osthole-loaded N-caprinoyl-N-trimethyl chitosan.

Figure 9 TEM image (A) of OST/CA-TMC2 micelles; AFM images (B) of OST/ CA-TMC2 micelles.

Abbreviations: AFM, atomic force microscopy; OST/CA-TMC2, osthole-loaded N-caprinoyl-N-trimethyl chitosan; TEM, transmission electron microscope.

Figure 10 In vitro release of OST in pH 5.0 PBS and pH 7.4 PBS; OST/CA-TMC2 micelles in pH 5.0 PBS and pH 7.4 PBS.

Abbreviations: OST, osthole; OST/CA-TMC2, osthole-loaded N-caprinoyl-N-trimethyl chitosan; PBS, phosphate-buffered saline.

Figure 11 Cytotoxic effects of OST against Hela and MCF-7 and OST/CA-TMC2 against Hela and MCF-7, respectively (n = 5).

Notes: *P < 0.05 vs OST group; **P < 0.01 vs OST group; or ^#P < 0.05 vs OST/ CA-TMC group; ^##P < 0.01 vs OST/CA-TMC group.

Abbreviations: OST, osthole; OST/CA-TMC2, osthole-loaded N-caprinoyl-N-trimethyl chitosan.

Table 3 Cell apoptosis of Hela and MCF-7 cells

Group	Cell population of Hela				Cell population of MCF-7
	%*	%**	%§	%†	%*	%**	%§	%†
Control	0.1	0.5	98.8	0.6	0.3	2.5	93.2	4.0
CA-TMC2 micelles	0.1	1.8	94.6	3.5	0.4	3.9	90.2	5.5
OST	1.0	2.8	60.2	36.0	6.7	12.6	62.5	18.1
OST/CA-TMC2 micelles	1.7	22.7	17.1	58.5	9.0	20.6	23.1	47.3

Notes:

* Unviable cell

** necrosis (or late apoptosis) cell

§ viable cell

† early apoptosis cell.

Abbreviations: CA-TMC, N-caprinoyl-N-trimethyl chitosan; OST, osthole; OST/CA-TMC, osthole-loaded N-caprinoyl-N-trimethyl chitosan.

Figure 12 Apoptosis evaluation after Hela and MCF-7 cells were treated with negative control (A), 420 μg • mL⁻¹ of CA-TMC2 micelles (B), 80 μg • mL⁻¹ of OST (C), and 420 μg • mL⁻¹ of OST/CA-TMC2 micelles including 80 μg • mL⁻¹ of OST (D).

Abbreviations: OST, osthole; OST/CA-TMC2, osthole-loaded N-caprinoyl-N-trimethyl chitosan.

Figure 13 CLSM images showing the uptake and subcellular localization of Hela (A) and MCF-7 (B).

Abbreviations: CLSM, confocal laser scanning microscope; OST/TITC-CA-TMC2, osthole loaded in N-caprinoyl-N-trimethyl chitosan with fluoredcein isothiocyanate marking.