Synergistic Fat-Reducing Effect of Deoxycholic Acid and Rhein in Lipid-Based Nanoparticles with Reduced Toxicity for Obesity Treatment

Ching-Yun Hsu,1,2,* Tse-Hung Huang,2– 6,* Zih-Chan Lin,7 Chih-Jung Chen,8– 10 Erica Hwang,11 Wei-Jhang Chen,12 Jia-You Fang2,12,13 1Department of Nutrition and Health Sciences, Chang Gung University of Science and Technology, Kweishan, Taoyuan, Taiwan; 2Research Center for Food and Cosmetic Safety and Research Center for Chinese Herbal Medicine, Chang Gung University of Science and Technology, Kweishan, Taoyuan, Taiwan; 3Department of Traditional Chinese Medicine, Chang Gung Memorial Hospital, Keelung, Taiwan; 4School of Traditional Chinese Medicine, Chang Gung University, Kweishan, Taoyuan, Taiwan; 5Department of Chemical Engineering and Graduate Institute of Biochemical Engineering, Ming Chi University of Technology, New Taipei City, Taiwan; 6Department of Traditional Chinese Medicine, Xiamen Chang Gung Memorial Hospital, Xiamen, People’s Republic of China; 7Chronic Diseases and Health Promotion Research Center, Chang Gung University of Science and Technology, Puzi, Chiayi, Taiwan; 8Department of Pathology and Laboratory Medicine, Taichung Veterans General Hospital, Taichung, Taiwan; 9School of Medicine, Chung Shan Medical University, Taichung, Taiwan; 10Department of Post-Baccalaureate Medicine, College of Medicine, National Chung Hsing University, Taichung, Taiwan; 11Department of Dermatology, Yale School of Medicine, Yale University, New Haven, CT, USA; 12Pharmaceutics Laboratory, Graduate Institute of Natural Products, Chang Gung University, Kweishan, Taoyuan, Taiwan; 13Department of Anesthesiology, Chang Gung Memorial Hospital, Linkou, Taiwan*These authors contributed equally to this workCorrespondence: Jia-You Fang, Pharmaceutics Laboratory, Graduate Institute of Natural Products, Chang Gung University, 259 Wen-Hwa 1st Road, Kweishan, Taoyuan, 333, Taiwan, Email fajy@mail.cgu.edu.twPurpose: Injectable deoxycholic acid (DA) has been approved for removing excess submental fat and is off-label for local adipose tissue reduction. Conventional DA injections fail to control fat reduction and generate severe adverse effects in adjacent non-adipose tissues. We designed squarticles as lipid-based nanoparticles for DA delivery to reduce fat accumulation.Methods: The liquid lipid phase of the squarticles was composed of squalene, which was previously reported to sequester the toxicity of overdosed drugs. Rhein, a natural anti-adipogenic compound, was incorporated into the squarticles for combined fat-lowering.Results: The squarticles had an average diameter of 93 nm and high rhein encapsulation (96%). The nanoparticles were easily internalized into mature adipocytes and were located in the lysosomes. DA induces adipocyte death via apoptosis and necrosis; however, nanoencapsulation can decrease cell death. Compared to free DA, squarticles showed superior mitigation of cytotoxicity against non-targeted cells (skin fibroblasts). Oil Red O staining indicated that squarticles loaded with DA or rhein alone inhibited lipid droplets by 42% and 17%, respectively. DA and rhein worked together in squarticles to further suppress fat accumulation by 50%. Dual administration of DA and rhein to the nanocarriers downregulated adipokines. The intraperitoneal administration of squarticles loaded with DA and rhein significantly decreased body weight, total cholesterol, and adipokine release. Histological analysis revealed that squarticles reduced adipocyte hypertrophy in the groin and epididymis by 11% and 53%, respectively. We examined the toxicity of the combination of DA+rhein in healthy rats that received a dose three-fold higher than that used in the pharmacological assessment. The survival rate of the overdosed DA+rhein increased from 50% to 100% after nanoencapsulation. Free compounds induce ascites, liver size reduction, AST/ALT elevation (1.5-fold), and potassium imbalance in rats. Nanoencapsulation significantly reduced these adverse effects.Conclusion: Our findings highlight the potential of squarticles for treating obesity. Keywords: deoxycholic acid, rhein, squarticle, lipid-based nanoparticle, obesity, adverse effect