Xiao-Man Huang,* Yan-Xiang Guo,* Qiu-Ling Pang, Xiao-Yi Yan, Hui Yan, Jing-Yi Li, Gan-Ling Tang, Hui-Xian Jiang, Hong-Liang Zhang

The First Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi, 530021, People’s Republic of China

*These authors contributed equally to this work

Correspondence: Hong-Liang Zhang, The First Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi, 530021, People’s Republic of China, Email 277749097@qq.com

Purpose: 2-Dodecyl-6-methoxy-2,5-diene-1,4-cyclohexanedione (DMDD), isolated from Averrhoa carambola L. root, has demonstrated the potential to reduce blood sugar levels. However, DMDD has poor solubility and bioavailability. This study aimed to formulate DMDD-loaded nanoparticles (DMDD-NPs) using chitosan crosslinked with sodium tripolyphosphate through the ionic crosslinking method and to investigate their effect on diabetic kidney disease (DKD) treatment by inhibiting the development of the epithelial-mesenchymal transition (EMT).
Methods: DMDD-NPs were prepared by ionic crosslinking with sodium tripolyphosphate, optimizing six factors that affect nanoparticle characteristics, including particle size and zeta potential. Encapsulation efficiency (EE) and drug loading rate (DL) were optimized using a Box-Behnken design. The structure and characteristics of DMDD-NPs, including size, EE, DL, and release rates, were analyzed. Cytotoxicity was assessed using the Cell Counting Kit-8 (CCK-8) assay, while the migration capacity of HK-2 cells was evaluated through scratch-wound assays. The expression of EMT-related markers (E-cadherin, Vimentin, and TGF-β 1) was assessed by qRT-PCR.
Results: The optimized formulation for DMDD-NPs was CS:TPP:DMDD = 10:3:3 (w), at pH 3.5, with 1.0 mg/mL of CS and stirring at 500 rpm for 30 min. In these conditions, the nanoparticles had a particle size of 320.37 ± 2.93 nm, an EE of 85.09 ± 1.43%, and a DL of 15.88 ± 0.51%. The DMDD-NPs exhibited a spherical shape, no leakage and minimal adhesion. The optimal freeze-drying protectant was a combination of 0.025% mannitol and 0.025% lactose. The drug release followed the Higuchi model. DMDD-NPs improved HK-2 cell proliferation at lower concentrations (< 24 μg/mL) and showed greater cell migration inhibition than DMDD. DMDD-NPs promoted E-cadherin expression and inhibited vimentin and TGF-β 1 expression, suggesting their potential role in preventing EMT for DKD treatment.

Keywords: DMDD, drug delivery system, nanoparticles, epithelial-mesenchymal transformation, diabetic kidney disease