Conferring liver selectivity to a thyromimetic using a novel nanoparticle increases therapeutic efficacy in a diet-induced obesity animal model

Abstract Optimization of metabolic regulation is a promising solution for many pathologies, including obesity, dyslipidemia, type 2 diabetes, and inflammatory liver disease. Synthetic thyroid hormone mimics–based regulation of metabolic balance in the liver showed promise but was hampered by the low biocompatibility and harmful effects on the extrahepatic axis. In this work, we show that specifically directing the thyromimetic to the liver utilizing a nanogel-based carrier substantially increased therapeutic efficacy in a diet-induced obesity mouse model, evidenced by the near-complete reversal of body weight gain, liver weight and inflammation, and cholesterol levels with no alteration in the thyroxine (T4) / thyroid stimulating hormone (TSH) axis. Mechanistically, the drug acts by binding to thyroid hormone receptor β (TRβ), a ligand-inducible transcription factor that interacts with thyroid hormone response elements and modulates target gene expression. The reverse cholesterol transport (RCT) pathway is specifically implicated in the observed therapeutic effect. Overall, the study demonstrates a unique approach to restoring metabolic regulation impacting obesity and related metabolic dysfunctions.

Table S3.List of genes examined by real-time PCR and the primer sequences used.Food intake was determined from total food consumption of mice every cage during a 24 h period so the statistical variation cannot be reported.

Figure S1 .Figure S3 .Figure S4 .
Figure S1.Characterization of PEG: PDS copolymers and ANG system.a, 1H nuclear magnetic resonance (1H-NMR) spectra and GPC trace of PEG: PDS random copolymers; b, Absorption spectra of pyridothione in UV-vis.The progress of cross-linking reaction and anionic ligand modification of nanogels was conveniently monitored by release of the pyridothione byproduct through tracing its characteristic absorption at 343 nm.

Figure S5 .
Figure S5.Quantitative fluorescence intensity of Cy7-NG in major organs from C57BL/6J mice receiving nanogels over time post-injection in ex vivo IVIS imaging study.Data are shown as the mean ± s.d. of n= 3 biologically independent mice per time point.Scale bar, red to blue, signal intensity high to low.Statistical significance was calculated via two-tailed Student's ttest.*P< 0.05; **P< 0.01; ***P< 0.001; ****P< 0.0001.

Figure S6 .
Figure S6.Effects of increasing dose of CGS-ANG, CGS 26214, T3 and ENG on total plasma triglyceride of normal mice.ENG is only shown at the 60 µg/kg/day, equivalent to the highest ANG in CGS-ANG dose.Results are expressed as fold change relative to the PBS vehicle treated control.Data are shown as mean ± s.e.m. (n= 6 biologically independent mice per group).Statistical significance was calculated via Ordinary one-way ANOVA with Tukey's multiple comparison test.

Figure S7 .
Figure S7.Relative mRNA expression of SR-B1, LDLR and SREBP-2 in livers from the PBS, CGS-ANG, CGS 26214, T3 and ENG treatment groups.ENG is only shown at the 60 µg/kg/day, equivalent to the highest ANG in CGS-ANG dose.All data are shown as mean ± s.e.m. (n= 6 biologically independent mice per group).Statistical significance was calculated via Ordinary one-way ANOVA with Tukey's multiple comparison test.

Figure S8 .
Figure S8.Schematic illustration of protocol for mild NASH disease establishment in C57BL/6J mice and preventing study of CGS-ANG.Twelve weeks after GAN diet feeding, mice were injected intraperitoneally daily by CGS 26214, CGS-ANG with three doses (dose 1,2,3 -CGS 10,20,60 μg/kg/day) and ENG for five weeks.CGS-ANG loaded with CGS 26214 at the dose that is the same as corresponding CGS 26214 groups.ENG contained the same dose of ANG with the highest dose CGS-ANG group.

Figure S21 .
Figure S21.Representative immunohistochemistry images and quantitative analysis of liver F4/80 and α-SMA in therapeutic study.Scale bar, 100 µm.Positive areas were quantified from three randomly chosen fields per liver section from individual mice using six mice per group.CGS-2 and CNG-2 were chosen as the representative in CGS and CGS-ANG groups.n= 6 biologically independent mice per group.Statistical significance was calculated via Ordinary one-way ANOVA with Tukey's multiple comparison test.*P< 0.05; **P< 0.01; ***P< 0.001; ****P< 0.0001 compared to NC control.

Figure S22 .
Figure S22.The protein levels of phospho-ERK1/2, ERK1/2, phospho-JNK, and JNK in the liver.The protein levels of phospho-ERK, ERK, phospho-JNK, and JNK in the livers of different treatment groups.The band densities for phospho-ERK, ERK, phospho-JNK, and JNK were first divided by GAPDH density to correct for very small loading differences.Then the levels of phospho-JNK/JNK and phospho-ERK/ERK were normalized to NC group.n= 8 biologically independent mice per group and every two mice were pooled as one sample.Cumulative densitometric analyses were performed from four independent gels.All data are shown as mean ± s.e.m.Statistical significance was calculated via Ordinary one-way ANOVA with Tukey's multiple comparison test.*P< 0.05; **P< 0.01; ***P< 0.001; ****P< 0.0001 compared to NC control.