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. 2023 Aug;28(2):142.
doi: 10.3892/mmr.2023.13029. Epub 2023 Jun 16.

A pH‑responsive hyaluronic acid nano‑vehicle co‑encapsulating doxorubicin and all‑trans retinoic acid for the inhibition of hepatic stellate cell‑induced tumor growth and metastasis

Qiao Lu #  1 , Xueyang Gong #  1 , Guangtao Jia #  1 , Jingliang Wu  2 , Siwei Liu  1 , Ke Song  1 , Guixiang Tian  1
Affiliations

A pH‑responsive hyaluronic acid nano‑vehicle co‑encapsulating doxorubicin and all‑trans retinoic acid for the inhibition of hepatic stellate cell‑induced tumor growth and metastasis

Qiao Lu et al. Mol Med Rep. 2023 Aug.

Abstract

All‑trans retinoic acid (ATRA) has been implicated in the differentiation of hepatic stellate cells (HSCs). In the present study, the liver‑targeting hyaluronic acid micelles (ADHG) were prepared for co‑delivery of ATRA and doxorubicin (DOX) to block the HSC‑hepatoma interrelation. To simulate the tumor microenvironment, an in vitro dual‑cell model and an in vivo co‑implantation mouse model were established for anticancer studies. The experimental methods involved the MTT assay, wound‑healing assay, cellular uptake, flow cytometry and and in vivo antitumor study. The results revealed that the HSCs in the research models notably promoted tumor proliferation and migration. Furthermore, ADHG were readily internalized by cancer cells and HSCs simultaneously, and widely distributed in cancer regions. The in vivo antitumor studies demonstrated that ADHG could notably decrease HSC activation and extracellular matrix deposition, as well as constrain tumor growth and metastasis. Therefore, ATRA could facilitate DOX‑induced anti‑proliferation and anti‑metastasis effects, and ADHG are a promising nano‑sized formulation for the combination therapy of hepatocellular carcinoma.

Keywords: all‑trans retinoic acid; anti‑hepatoma; combination therapy; hepatic stellate cells.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1.
Figure 1.
Illustration and characteristics of drug-loaded nanoparticles. (A) Design of the combination therapy. Transmission electron microscope images and particle sizes of (B) DOX-sHA-GA and (C) ATRA/DOX-sHA-GA (scale bar, 200 nm). (D and E) Stability of ADHG under physiological conditions within 7 days. (D) DLS and (E) Zeta potential. ADHG, ATRA/DOX-sHA-GA; DOX, doxorubicin; sHA, sulfated hyaluronic acid; GA, glycyrrhetinic acid; ATRA, all-trans retinoic acid; DLS, dynamic light scattering.
Figure 2.
Figure 2.
Cellular uptake and cytotoxicity. (A) Two research models used in the present study. Images of (B) Huh-7 cells and (C) the dual-cell model. White arrows indicate the fluorescence intensity of Huh-7 cells. (D) Flow cytometry assay and (E) its quantitative analysis. Cytotoxicity of (F) Huh-7 cells alone and (G) the dual-cell model. (H) Live/dead staining test (scale bar, 100 µm). *P<0.05, **P<0.01 and ***P<0.001. DHG, DOX-sHA-GA; DH, DOX-sHA; DOX, doxorubicin; CFSE, 5(6)-carboxyfluorescein diacetate succinimidyl ester; ATRA, all-trans retinoic acid; ADH, ATRA/DOX-sHA; ADHG, ATRA/DOX-sHA-GA; sHA, sulfated hyaluronic acid.
Figure 3.
Figure 3.
Anti-migration effect of ADHG. Wound-healing assay using (A) Huh-7 cells alone and (B) the dual-cell research model (scale bar, 100 µm). Migration rates of (C) Huh-7 cells alone and (D) the dual-cell model. The experiment was performed in triplicate. *P<0.05, **P<0.01 and ***P<0.001. ATRA, all-trans retinoic acid; ADH, ATRA/DOX-sHA; ADHG, ATRA/DOX-sHA-GA; DOX, doxorubicin; DH, DOX-sHA; sHA, sulfated hyaluronic acid.
Figure 4.
Figure 4.
In vivo drug distribution in subcutaneous tumor-bearing mice. DiR fluorescence imaging of DiR, DH/DiR and DHG/DiR in 0–48 h. DiR, 1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine iodide; DH, doxorubicin-sulfated hyaluronic acid; DHG, doxorubicin-sulfated hyaluronic acid-glycyrrhetinic acid.
Figure 5.
Figure 5.
Evaluation of anti-hepatoma activity in H22/m-HSC-bearing mice. (A) Treatment schedule. (B) Tumor images. (C) Tumor volumes. (D) Tumor growth inhibition rates. (E) H&E and (F) Masson staining assays. Immunohistochemical assays of (G) α-SMA and (H) CD31 proteins, with red arrows indicating the tumor vessels (scale bars, 50 µm). *P<0.05 and ***P<0.001. m-HSC, mouse hepatic stellate cells; SMA, smooth muscle actin; ATRA, all-trans retinoic acid; ADH, ATRA/DOX-sHA; ADHG, ATRA/DOX-sHA-GA; DOX, doxorubicin; DH, DOX-sHA; sHA, sulfated hyaluronic acid.
Figure 6.
Figure 6.
Evaluation of the anti-metastatic effect of ADHG. (A) Model establishment and drug administration. (B) Mice weight over time. (C) Quantitative analysis of metastatic nodules. (D) Lung images; circles indicate transfer areas of tumor metastasis. (E) H&E analysis (magnification, ×200). *P<0.05, **P<0.01 and ***P<0.001. ATRA, all-trans retinoic acid; ADH, ATRA/DOX-sHA; ADHG, ATRA/DOX-sHA-GA; DOX, doxorubicin; DH, DOX-sHA; sHA, sulfated hyaluronic acid.
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