Molecular medicine reports 19: 133-142, 2019

Doxorubicin‑loaded dual‑functional hyaluronic 
acid nanoparticles: Preparation, characterization 
and antitumor efficacy in vitro and in vivo
GUIXIANG TIAN
1,2*
, XIUE SUN
2*
, JINGKUN BAI
1
, JINHUA DONG
1,3
,
BO ZHANG
4
, ZHIQIN GAO
1,3
and JINGLIANG WU
1,3
1
School of Bioscience and Technology; 
2
Department of Psychology; 
3
Key Laboratory of Biological Medicine in Universities of Shandong Province; 
4
School of Pharmacy, Weifang Medical University, Weifang, Shandong 261053, P.R. China
Received April 2, 2018; Accepted October 10, 2018
DOI: 10.3892/mmr.2018.9687
Correspondence to:
Ms. Guixiang Tian, Department of Psychology, 
Weifang Medical University, 7166 Baotong West Street, Weifang, 
Shandong 261053, P.R. China
E-mail: [e-mail]
Dr Jingliang Wu, School of Bioscience and Technology, Weifang 
Medical University, 7166 Baotong West Street, Weifang, 
Shandong 261053, P.R. China
E-mail: [e-mail]
*
Contributed equally
Key words:
hyaluronic acid, nanoparticles, liver-targeting, 
pH-responsive

---

TIAN et al: DUAL-FUNCTIONAL HYALURONIC ACID NANOPARTICLES
134
Materials and methods
Materials. Hyaluronic acid (HA) (MW, 80 kDa) was 
purchased from Bloomage Freda Biopharm Co., Ltd. (Jinan, 
China). L-histidine (His) was purchased from Sinopharm 
Chemical Reagent Co., Ltd. (Shanghai, China). Glycyrrhetinic 
acid (GA) was acquired from Meheco Tianshan Pharm Co., 
Ltd. (Beijing, China). DOX·HCl was purchased from Shanghai 
Sangon Biomart Co., Ltd. (Shanghai, China).
4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium 
chloride (DMT-MM), pyrene and MTT was procured from 
Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). RPMI-1640 
medium was purchased from Beijing BioDee Biotechnology Co., 
Ltd. (Beijing, China). All chemicals were of analytical grade.
Cell cultures. Human hepatic cell line (HepG2) was obtained 
from the China Center for Type Culture Collection (Wuhan, 
China), while murine HCC cells (H22) were gifted by the 
Institute of Immunopharmacology and Immunotherapy of 
Shandong University (Jinan, China). Both cell lines were 
cultured in RPMI-1640 medium, supplemented with 10% fetal 
bovine serum (FBS), 1% penicillin and 1% streptomycin at 
37˚C in an environment containing 5% CO
2
.
Animals. Female BALB/c mice (weight: 18±2 g) were supplied 
by the Experimental Animal Center of WeiFang Medical 
University (Weifang, China). In total 36 mice were used for 
in vivo imaging and antitumor efficacy experiments. The 
animals were fed at 25±2˚C in the institutional animal house 
facility (relative humidity: 40-70%, 12-h/d light dark cycle), 
with a standard diet and allowed water ad libitum.
Synthesis of GHH copolymers. GHH copolymers were 
synthesized through a two-step reaction. First, GA solution in 
methanol was activated to form an active ester in the pres-
ence of DMT-MM. The active ester solution was evaporated 
to remove methanol, and slowly added to an ethylene diamine 
solution under stirring at room temperature for 24 h. Then, the 
diamine‑modified GA (GA‑NH
2
) was obtained after purifica-
tion by column chromatography. The GA-HA conjugate was 
synthesized by the chemical modification of GA–NH
2
to the 
backbone of HA (70 kDa). Second, the GA-HA conjugate was 
dissolved in formylamine before DMT-MM was slowly added. 
Then, His was slowly added to the GA-HA solution, followed 
by stirring at room temperature for 24 h. After filtration, the 
solution was freeze-dried to obtain GHH copolymers. The 
chemical structures of the GHH conjugates were determined 
by 
1
H NMR (JNM ECP-600, JEOL, Japan) by dissolving the 
conjugate in D
2
O.