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International Journal of Nanomedicine Volume 14, 2019 - Issue
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Original Research

Liver-Targeting and pH-Sensitive Sulfated Hyaluronic Acid Mixed Micelles for Hepatoma Therapy

Zhi-peng Li1 School of Bioscience and Technology, Weifang Medical University, Weifang, Shandong, People’s Republic of ChinaORCID Iconhttps://orcid.org/0000-0001-8154-6221
ORCID Icon,
Gui-xiang Tian1 School of Bioscience and Technology, Weifang Medical University, Weifang, Shandong, People’s Republic of China
,
Hong Jiang1 School of Bioscience and Technology, Weifang Medical University, Weifang, Shandong, People’s Republic of ChinaORCID Iconhttps://orcid.org/0000-0002-6063-7324
ORCID Icon,
Rui-yan Pan2 School of Pharmacy, Weifang Medical University, Weifang, Shandong, People’s Republic of China
,
Bo Lian1 School of Bioscience and Technology, Weifang Medical University, Weifang, Shandong, People’s Republic of China
,
Min Wang1 School of Bioscience and Technology, Weifang Medical University, Weifang, Shandong, People’s Republic of China
,
Zhi-qin Gao1 School of Bioscience and Technology, Weifang Medical University, Weifang, Shandong, People’s Republic of China
,
Bo Zhang2 School of Pharmacy, Weifang Medical University, Weifang, Shandong, People’s Republic of ChinaCorrespondence[email protected]
&
Jing-liang Wu1 School of Bioscience and Technology, Weifang Medical University, Weifang, Shandong, People’s Republic of ChinaCorrespondence[email protected]
ORCID Iconhttps://orcid.org/0000-0001-6962-2842
ORCID Icon show all
Pages 9437-9452 | Published online: 02 Dec 2019

Figures & data

Figure 1 Illustration of the formation, uptake by tumor cells, and drug release of sHA-DOX/HA-GA mixed micelles.

Abbreviations: HA, hyaluronic acid; sHA, sulfated hyaluronic acid; DOX, doxorubicin; GA, glycyrrhetinic acid; ADH, adipic acid dihydrazide.

Figure 1 Illustration of the formation, uptake by tumor cells, and drug release of sHA-DOX/HA-GA mixed micelles.Abbreviations: HA, hyaluronic acid; sHA, sulfated hyaluronic acid; DOX, doxorubicin; GA, glycyrrhetinic acid; ADH, adipic acid dihydrazide.

Figure 2 Synthesis of sHA-DOX Polymers.

Note: 0 < x ≤ 1, otherwise –OH group.

Abbreviations: HA, hyaluronic acid; sHA, sulfated hyaluronic acid; HOBt, 1-hydroxybenzotriazole; EDC, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; DOX·HCl, doxorubicin·HCl; DOX, doxorubicin; TEA, triethylamine; rt, room temperature.

Figure 2 Synthesis of sHA-DOX Polymers.Note: 0 < x ≤ 1, otherwise –OH group.Abbreviations: HA, hyaluronic acid; sHA, sulfated hyaluronic acid; HOBt, 1-hydroxybenzotriazole; EDC, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; DOX·HCl, doxorubicin·HCl; DOX, doxorubicin; TEA, triethylamine; rt, room temperature.

Figure 3 (A) IR spectra of HA, sHA and sHA-DOX Polymers, (B) 1H NMR spectra of HA, sHA, DOX and sHA-DOX Polymers in D2O.

Abbreviations: HA, hyaluronic acid; sHA, sulfated hyaluronic acid; DOX, doxorubicin; IR, infrared spectroscopy; NMR, nuclear magnetic resonance.

Figure 3 (A) IR spectra of HA, sHA and sHA-DOX Polymers, (B) 1H NMR spectra of HA, sHA, DOX and sHA-DOX Polymers in D2O.Abbreviations: HA, hyaluronic acid; sHA, sulfated hyaluronic acid; DOX, doxorubicin; IR, infrared spectroscopy; NMR, nuclear magnetic resonance.

Figure 4 The stability of sHA-DOX/HA-GA mixed micelles.

Notes: The particle size distribution of sHA-DOX (A), HA-GA (B), sHA-DOX/HA-GA (C); the particle size (D) and zeta potential (E) of sHA-DOX/HA-GA mixed micelles for 2 weeks in PBS; the particle size changes of sHA-DOX/HA-GA mixed micelles for 7 days in RPMI 1640 medium (F).

Abbreviations: HA, hyaluronic acid; sHA, sulfated hyaluronic acid; DOX, doxorubicin; GA, glycyrrhetinic acid; DLS, dynamic light scattering.

Figure 4 The stability of sHA-DOX/HA-GA mixed micelles.Notes: The particle size distribution of sHA-DOX (A), HA-GA (B), sHA-DOX/HA-GA (C); the particle size (D) and zeta potential (E) of sHA-DOX/HA-GA mixed micelles for 2 weeks in PBS; the particle size changes of sHA-DOX/HA-GA mixed micelles for 7 days in RPMI 1640 medium (F).Abbreviations: HA, hyaluronic acid; sHA, sulfated hyaluronic acid; DOX, doxorubicin; GA, glycyrrhetinic acid; DLS, dynamic light scattering.

Table 1 Characterization of Different Micelles

Figure 5 pH-responsive size behaviors of mixed micelles.

Notes: (A) Particle size variation of mixed micelles at different pH values within 8 hrs. (B) TEM image of mixed micelles at pH 7.4. (C) TEM image of mixed micelles at pH 5.5 after 4 hrs.

Figure 5 pH-responsive size behaviors of mixed micelles.Notes: (A) Particle size variation of mixed micelles at different pH values within 8 hrs. (B) TEM image of mixed micelles at pH 7.4. (C) TEM image of mixed micelles at pH 5.5 after 4 hrs.

Figure 6 In vitro release of DOX from sHA-DOX/HA-GA mixed micelles.

Notes: Release medium: 50 mL of PBS (pH 7.4, 6.8, and 5.5) containing 0.5% Tween 80; Release condition: 37°C, 100 rpm, under dark condition. **P<0.01. Abbreviations: HA, hyaluronic acid; sHA, sulfated hyaluronic acid; DOX, doxorubicin; GA, glycyrrhetinic acid.

Figure 6 In vitro release of DOX from sHA-DOX/HA-GA mixed micelles.Notes: Release medium: 50 mL of PBS (pH 7.4, 6.8, and 5.5) containing 0.5% Tween 80; Release condition: 37°C, 100 rpm, under dark condition. **P<0.01. Abbreviations: HA, hyaluronic acid; sHA, sulfated hyaluronic acid; DOX, doxorubicin; GA, glycyrrhetinic acid.

Figure 7 Confocal images of HepG2 cells incubated with sHA-DOX (A) and sHA-DOX/HA-GA (B); HeLa cells incubated with sHA-DOX (C) and sHA-DOX/HA-GA (D).

Note: DAPI (blue), DOX (red) and a merge of two images were simultaneously presented.

Abbreviations: HA, hyaluronic acid; sHA, sulfated hyaluronic acid; DAPI, 4ʹ,6-diamidino-2-phenylindole; DOX, doxorubicin; GA, glycyrrhetinic acid.

Figure 7 Confocal images of HepG2 cells incubated with sHA-DOX (A) and sHA-DOX/HA-GA (B); HeLa cells incubated with sHA-DOX (C) and sHA-DOX/HA-GA (D).Note: DAPI (blue), DOX (red) and a merge of two images were simultaneously presented.Abbreviations: HA, hyaluronic acid; sHA, sulfated hyaluronic acid; DAPI, 4ʹ,6-diamidino-2-phenylindole; DOX, doxorubicin; GA, glycyrrhetinic acid.

Figure 8 Cytotoxicity of HepG2 (A) and HeLa cells (B) incubated with different concentrations of free DOX, HA-DOX, sHA-DOX and sHA-DOX/HA-GA for 24 hrs.

Notes: *P<0.05, free DOX vs sHA-DOX/HA-GA group, sHA-DOX vs sHA-DOX/HA-GA group; **P<0.01, free DOX vs sHA-DOX/HA-GA group; n.s., nonsignificant.

Abbreviations: HA, hyaluronic acid; sHA, sulfated hyaluronic acid; DOX, doxorubicin; GA, glycyrrhetinic acid.

Figure 8 Cytotoxicity of HepG2 (A) and HeLa cells (B) incubated with different concentrations of free DOX, HA-DOX, sHA-DOX and sHA-DOX/HA-GA for 24 hrs.Notes: *P<0.05, free DOX vs sHA-DOX/HA-GA group, sHA-DOX vs sHA-DOX/HA-GA group; **P<0.01, free DOX vs sHA-DOX/HA-GA group; n.s., nonsignificant.Abbreviations: HA, hyaluronic acid; sHA, sulfated hyaluronic acid; DOX, doxorubicin; GA, glycyrrhetinic acid.

Figure 9 Effect of DOX, HA-DOX, sHA, sHA-DOX, and sHA-DOX/HA-GA on cell migration (A). Cell migration rate from 0 to 24 hrs was calculated by ImageJ (B).

Notes: Data represent mean ± SD (n=3); Control cells treated with serum-free medium. **P<0.01, statistically significant difference between free DOX and sHA group; ***P<0.001, statistically significant difference between free DOX and sHA-DOX group, between free DOX and sHA-DOX/HA-GA group.

Abbreviations: HA, hyaluronic acid; sHA, sulfated hyaluronic acid; DOX, doxorubicin; GA, glycyrrhetinic acid.

Figure 9 Effect of DOX, HA-DOX, sHA, sHA-DOX, and sHA-DOX/HA-GA on cell migration (A). Cell migration rate from 0 to 24 hrs was calculated by ImageJ (B).Notes: Data represent mean ± SD (n=3); Control cells treated with serum-free medium. **P<0.01, statistically significant difference between free DOX and sHA group; ***P<0.001, statistically significant difference between free DOX and sHA-DOX group, between free DOX and sHA-DOX/HA-GA group.Abbreviations: HA, hyaluronic acid; sHA, sulfated hyaluronic acid; DOX, doxorubicin; GA, glycyrrhetinic acid.

Figure 10 In vivo biodistribution of sHA-DOX/HA-GA mixed micelles.

Notes: (A) Mice treated with DiR solution via tail vein injection; (B) mice treated with DiR-loaded sHA-DOX/HA-GA mixed micelles via tail vein injection. Red circles indicate tumor sites.

Abbreviations: HA, hyaluronic acid; sHA, sulfated hyaluronic acid; DOX, doxorubicin; GA, glycyrrhetinic acid.

Figure 10 In vivo biodistribution of sHA-DOX/HA-GA mixed micelles.Notes: (A) Mice treated with DiR solution via tail vein injection; (B) mice treated with DiR-loaded sHA-DOX/HA-GA mixed micelles via tail vein injection. Red circles indicate tumor sites.Abbreviations: HA, hyaluronic acid; sHA, sulfated hyaluronic acid; DOX, doxorubicin; GA, glycyrrhetinic acid.

Figure 11 In vivo antitumor efficacy of DOX, HA-DOX, sHA-DOX, and sHA-DOX/HA-GA on H22 tumor-bearing mice. (A) The changes in body weight of female BALB/c mice; (B) Tumor growth curve; (C) The images of excised tumors; (D) Tumor growth-inhibition rate. The error bars represent standard deviation (n=4).

Notes: Saline, mice treated with normal saline via tail vein injection. ***P<0.001, statistically significant difference between free DOX and sHA-DOX group, between free DOX and sHA-DOX/HA-GA group; ##P<0.01, statistically significant difference between free DOX and sHA-DOX group; ###P<0.001, statistically significant difference between free DOX and sHA-DOX/HA-GA group.

Abbreviations: HA, hyaluronic acid; sHA, sulfated hyaluronic acid; DOX, doxorubicin; GA, glycyrrhetinic acid.

Figure 11 In vivo antitumor efficacy of DOX, HA-DOX, sHA-DOX, and sHA-DOX/HA-GA on H22 tumor-bearing mice. (A) The changes in body weight of female BALB/c mice; (B) Tumor growth curve; (C) The images of excised tumors; (D) Tumor growth-inhibition rate. The error bars represent standard deviation (n=4).Notes: Saline, mice treated with normal saline via tail vein injection. ***P<0.001, statistically significant difference between free DOX and sHA-DOX group, between free DOX and sHA-DOX/HA-GA group; ##P<0.01, statistically significant difference between free DOX and sHA-DOX group; ###P<0.001, statistically significant difference between free DOX and sHA-DOX/HA-GA group.Abbreviations: HA, hyaluronic acid; sHA, sulfated hyaluronic acid; DOX, doxorubicin; GA, glycyrrhetinic acid.

Figure 12 Images of H&E assays for tumors (A), hearts (B), and MVD determination by immunohistochemistry on formaldehyde-fixed tumors (C) after treatment with saline, free DOX, HA-DOX, sHA-DOX and sHA-DOX/HA-GA for 2 weeks. For all images, magnification is 400 ×.

Notes: Saline, mice treated with normal saline via tail vein injection. Green arrows indicate tumor necrotic area in (A), myocardial injury area in (B). Red arrowheads indicate the tumor microvessels in (C).

Abbreviations: HA, hyaluronic acid; sHA, sulfated hyaluronic acid; DOX, doxorubicin; GA, glycyrrhetinic acid; H&E, Hematoxylin-eosin; MVD, microvessel density.

Figure 12 Images of H&E assays for tumors (A), hearts (B), and MVD determination by immunohistochemistry on formaldehyde-fixed tumors (C) after treatment with saline, free DOX, HA-DOX, sHA-DOX and sHA-DOX/HA-GA for 2 weeks. For all images, magnification is 400 ×.Notes: Saline, mice treated with normal saline via tail vein injection. Green arrows indicate tumor necrotic area in (A), myocardial injury area in (B). Red arrowheads indicate the tumor microvessels in (C).Abbreviations: HA, hyaluronic acid; sHA, sulfated hyaluronic acid; DOX, doxorubicin; GA, glycyrrhetinic acid; H&E, Hematoxylin-eosin; MVD, microvessel density.

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