(M1130-05-32) Targeted Transport of Active Pharmaceutical Ingredients into Adipose Tissue and Liver

Purpose: The rising prevalence of obesity and non-alcoholic fatty liver disease (NAFLD) and the high association between obesity and NAFLD have prompted the urgent need for effective pharmacological interventions ^1–3. Triiodothyronine (T3), a biologically active thyroid hormone, shows promise as a therapeutic agent for combating obesity and NAFLD due to its ability to activate brown adipose tissue (BAT) and regulate liver lipid metabolism^4,5. Here, we established a delivery system for the targeted transport of T3 to BAT and the liver, aiming to activate BAT and alleviate diet-induced hepatic steatosis.

Methods: Triglyceride-rich lipoproteins (TRLs), also known as artificial chylomicrons, were prepared as delivery carriers to encapsulate T3 using the thin-film evaporation-sonication method ⁶ (Fig. 1A). The characteristics of T3-loaded TRLs (T3-TRLs), including particle size, polydispersity index (PDI), zeta potential, and encapsulation efficiency, were assessed using Zetasizer and high-performance liquid chromatography (HPLC). To investigate the impact of T3 on BAT activation, wild-type (WT) C57BL/6 male mice at the age of 8 weeks subjected to cold exposure were intravenously administered T3-TRLs for one week (Fig. 1B). Moreover, a 2-week treatment with T3-TRLs was performed on diet-induced obese WT mice to assess the effects of T3-TRLs on glucose tolerance improvement and attenuation of hepatic steatosis (Fig. 2A).

Results: T3-TRLs were characterized by particle size (275.1 ± 7.8 nm), PDI (0.22 ± 0.02), zeta potential (-10.8 ± 0.4 mV), and encapsulation efficiency (40.8 ± 1.8%). Treatment with T3-TRLs resulted in the enhancement of the thermogenic process within BAT, as increased by the upregulation of Ucp1 and Dio2 gene expression by 3.4 ± 0.9-fold and 4.7 ± 1.2-fold, respectively (Fig. 1C). Histological analysis further revealed an increase in multilocular lipid droplets and UCP1 staining in BAT compared to the control group. T3-TRLs treatment also led to a significant increase in the expression of hepatic genes associated with liver metabolism, including Dio1, Cyp7α1, Ldlr, Pparα, and Cpt1α (Fig. 1D and E). Additionally, glucose tolerance in the obese mouse model was improved following treatments with T3-TRLs (Fig. 2B and C). Hepatic gene expression demonstrated an upregulation of Glut4, Ldlr, and Lipc in response to T3-TRLs treatments, while the expression of Dio3 in the brain increased only in the T3-treated group, indicating T3-TRLs delivered T3 to the liver but not in the brain due to the blood-brain barrier (Fig. 2D and E). Notably, T3-TRLs treatments reduced the steatosis in the liver by 48.1 ± 8.1% as quantified in the histological analysis of the liver (Fig. 2F), as well as the hepatic triglyceride content following treatment with T3-TRLs and T3 was dramatically reduced to 48.7 ± 7.8% and 65.7 ± 11.9%, respectively (Fig. 2G).

Conclusion: T3-TRLs can target T3 to BAT in mice after cold exposure and increase BAT UCP1 expression. Importantly, T3-TRLs treatment demonstrates promising improvements in glucose tolerance and reduction of hepatic steatosis in obese mice, providing a potential therapeutic strategy in obesity-related conditions.

References: 1. Fabbrini, E., Sullivan, S. & Klein, S. Obesity and Nonalcoholic Fatty Liver Disease: Biochemical, Metabolic and Clinical Implications. Hepatology 51, 679–689 (2010).
2. Mitchell, N., Catenacci, V., Wyatt, H. R. & Hill, J. O. OBESITY: OVERVIEW OF AN EPIDEMIC. Psychiatr Clin North Am 34, 717–732 (2011).
3. Riazi, K. et al. The prevalence and incidence of NAFLD worldwide: a systematic review and meta-analysis. The Lancet Gastroenterology & Hepatology 7, 851–861 (2022).
4. Yau, W. W. et al. Thyroid hormone (T3) stimulates brown adipose tissue activation via mitochondrial biogenesis and MTOR-mediated mitophagy. Autophagy 15, 131–150 (2019).
5. Sinha, R. A., Singh, B. K. & Yen, P. M. Direct effects of thyroid hormones on hepatic lipid metabolism. Nat Rev Endocrinol 14, 259–269 (2018).
6. Hildebrand, S. et al. Quantification of Lipoprotein Uptake in Vivo Using Magnetic Particle Imaging and Spectroscopy. ACS Nano 15, 434–446 (2021).

Acknowledgements: Zheming Niu was supported by the Bonn International Graduate School of Drug Sciences (BIGS DrugS), University of Bonn, Germany. Staffan Hildebrand thanks the DFG (FOR917, 335447717/SFB1328, and 397484323/SFB TRR 259/1) and DAAD (57172123) funding. Zheming Niu thanks the Argelander International Conference Grant, University of Bonn for travel funding. The approval for animal experiments has been granted by the Landesamt für Natur, Umwelt und Verbraucherschutz (LANUV), located in Nordrhein-Westfalen, Germany, under the approval number 81-02.04.2019.A099.

Fig. 1 Targeting T3-TRLs to BAT. (A) The structure of T3-TRLs, (B) The treatment plan, (C) Ucp1 and Dio2 expression in BAT after treatments with Vehicle-TRLs and T3-TRLs, (D) Dio1 and Pparα expression in the liver after treatments with Vehicle-TRLs and T3-TRLs, (E) Ldlr, Cyp7α1, and Cpt1α expression in the liver for bile acid synthesis and hepatic lipolysis after treatments. n = 3 - 4. The data are presented as means ± s.e.m and analyzed using a t-test. * p < 0.05.

Fig. 2 Chronic treatment with T3-TRLs during DIO. (A) The treatment plan, (B) Glucose tolerance test (GTT) in DIO mice before and after treatments with T3-TRLs, (C) Respective area under the curve (AUC) of GTT measurement after treatments, (D) Glut4, Ldlr, and Lipc expression in the liver after treatments with Vehicle-TRLs and T3-TRLs, (E) Dio3 expression in the brain after treatments, (F) Quantification of hepatic steatosis after treatments, (G) Hepatic triglyceride content. n = 5. The data are presented as means ± s.e.m and analyzed using a t-test or ANOVA. * p < 0.05, ** p < 0.01, **** p < 0.0001.