Fabrication and characterization of nuclear localization signal-conjugated glycol chitosan micelles for improving the nuclear delivery of doxorubicin

Figures & data

Table 1 Physicochemical properties of drug-free and drug-loaded micelles

Sample	DOX/carriera	Size (nm)b	PI	Zeta potential (mV)	LC (%)	EE (%)
CHGC	–	214 ± 25.6	0.226 ± 0.073	13.11 ± 0.72	–	–
NCHGC	–	205 ± 18.7	0.233 ± 0.068	11.87 ± 0. 89	–	–
DCHGC	1.5/10	246 ± 22.3	0.219 ± 0.076	11.93 ± 0.55	11.5 ± 0.85	86.7± 6.41
DNCHGC	1.5/10	248 ± 20.1	0.241 ± 0.083	19.05 ± 1.22	10.1 ± 0.79	75.0 ± 6.52

Notes:

a The ratio of DOX to carrier, based on feed amount (mg/mg);

b measured by dynamic light scattering and described in terms of intensity distributions. The results represent the means ± standard deviation (n = 3).

Abbreviations: DOX, doxorubicin; PI, polydispersity index; LC, loading content; EE, entrapment efficiency; CHGC, cholesterol-modified glycol chitosan; DCHGC, doxorubicin-loaded cholesterol-modified glycol chitosan; NCHGC, nuclear localization signal-conjugated cholesterol-modified glycol chitosan; DNCHGC, doxorubicin-loaded nuclear localization signal-conjugated cholesterol-modified glycol chitosan.

Figure 1 Synthetic scheme of NCHGC.

Abbreviations: EDC, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride; CHGC, cholesterol-modified glycol chitosan; SMCC, N-succinimidyl 4-(maleimidomethyl) cyclohexane carboxylate; NCHGC, nuclear localization signal-conjugated cholesterol-modified glycol chitosan; NLS, nuclear localization signal.

Figure 2 Proton nuclear magnetic resonance spectra of (A) glycol chitosan, (B) cholesterol-modified glycol chitosan (CHGC), (C) nuclear localization signal (NLS) (Ac-CGYGPKKKRKVGG), and (D) NLS-conjugated cholesterol-modified glycol chitosan (NCHGC).

Figure 3 Plots of the intensity ratio I₃₃₈/I₃₃₃ vs log C for nuclear localization signal-conjugated cholesterol-modified glycol chitosan with various compositions.

Figure 4 Schematic illustration of the preparation procedure of doxorubicin-loaded nuclear localization signal-conjugated cholesterol-modified glycol chitosan micelles by an emulsion/solvent evaporation method.

Abbreviation: NLS, nuclear localization signal (Ac-CGYGPKKKRKVGG).

Figure 5 Transmission electron microscopy image of doxorubicin-loaded nuclear localization signal-conjugated cholesterol-modified glycol chitosan micelles (×20,000).

Figure 6 Release profiles of doxorubicin (DOX) from doxorubicin-loaded cholesterol-modified glycol chitosan (DCHGC) and doxorubicin-loaded nuclear localization signal-conjugated cholesterol-modified glycol chitosan (DNCHGC) micelles at 37°C in phosphate-buffered saline containing 10% fetal bovine serum at pH 7.4.

Figure 7 Flow cytometric analysis of HeLa cells incubated with free doxorubicin (DOX), doxorubicin-loaded cholesterol-modified glycol chitosan (DCHGC) and doxorubicin-loaded nuclear localization signal-conjugated cholesterol-modified glycol chitosan (DNCHGC) micelles for 2 hours.

Figure 8 Confocal laser scanning microscopy images of HeLa cells after incubation with (A) free doxorubicin (DOX), (B) doxorubicin-loaded cholesterol-modified glycol chitosan micelles and (C) doxorubicin-loaded nuclear localization signal-conjugated cholesterol-modified glycol chitosan micelles for 2 hours. The red DOX fluorescence was observed in (A1–C1). The nuclei were stained with Hoechst 33342 in (A2–C2), and the merged photos were presented in (A3–C3).

Figure 9 In vitro cytotoxicity of blank micelles and various doxorubicin (DOX) formulations against HepG2 and HeLa cells after 48 hours of incubation. (A) Cholesterol-modified glycol chitosan (CHGC) and nuclear localization signal-conjugated CHGC (NCHGC) micelles in HepG2 cells; (B) free DOX, doxorubicin-loaded CHGC (DCHGC) and doxorubicin-loaded nuclear localization signal-conjugated CHGC (DNCHGC) micelles in HepG2 cells; (C) CHGC and NCHGC micelles in HeLa cells; and (D) free DOX, DCHGC and DNCHGC micelles in HeLa cells.