Simvastatin-Loaded Polymeric Nanoparticles: Targeting Inflammatory Macrophages for Local Adipose Tissue Browning in Obesity Treatment

Introduction: Currently, obesity is an ongoing global health crisis. The American Medical Association classified obesity as a disease in 2013 and identified it as one of the most severe public health issues and the global epidemic of the 21st century [1]. In obesity, fat is stored in excess in the body due to an imbalance in the intake and expenditure of energy. People who suffer from this chronic disease are at a major risk of developing serious complications, such as hepatic steatosis, metabolic syndrome, type 2 diabetes, cardiovascular disease, and cancer [2], all of which severely reduce their life expectancy [3]. Several approaches have been used to treat obesity, such as diet calorie restriction by diet programs, physical exercise, anti-adipogenesis medications (like prostaglandin E2 analog, rosiglitazone, and L-carnitine), pharmacological therapy, liposuction, as well as bariatric surgery [4, 5]. Exercise and dieting can effectively balance energy consumption and absorption, but clinical interventions are strongly recommended when reversing severe obesity. Many aggressive anti-obesity therapeutic treatments have high risks [6] and serious unintended side effects on human organs like the liver, gastrointestinal, and kidney [7, 8]. So, we expected that an adipocyte-specific, minimally invasive approach to obesity would inspire significant enthusiasm.

Materials and

Methods: Sim-PLGA NPs were formulated at a Sim:PLGA, with a mass ratio of 1:4 by the emulsification solvent evaporation technique. PLGA and Sim were first dissolved in dichloromethane, mixed, and stirred. A 1% (w/v) PVA aqueous solution was dropped into the above mixture while sonicated for 20 minutes. In the final stage, the pellet was dispersed in DI and followed by freeze-drying for 24 h. The encapsulation efficiency of Sim was measured by HPLC. To evaluate Sim release characteristics, we investigated in vitro release profiles of Sim from Sim-PLGA NPs under neutral (1×PBS buffer [pH=7.4]) conditions. The immunomodulation of Sim-PLGA NPs was studied in vitro and in vivo using bone marrow-derived macrophages (BMDM) and dendritic cells (BMDC) and assessed for inflammatory cytokine secretions (IL-2, IFN-ɣ, IL-6, TNF-α), and upregulation of co-stimulatory molecules (CD206, CD80/86) using flow cytometry. To study the effect of Sim-PLGA NPs on the modulation of macrophages, bone marrow harvested from mice was cultured for eight days with cytokines to differentiate the monocyte to macrophages and then treated with LPS to activate macrophages and polarize them to M1-like macrophages. High Fat Diet-Induced Obesity Model (HFD) was induced in C57BL/6 mice by being fed high-fat or high-density diets to mice for 8 weeks. Mice (n=16) were then injected subcutaneously with Sim-PLGA NPs, free Sim, or PBS on days 4, 11, and 18. Mice were monitored daily and weighed. Six weeks after the treatments, mice were euthanized, and tissue was collected and studied.

Results, Conclusions, and Discussions: Scanning electron microscopy (SEM) showed the spherical structures of synthesized Sim-PLGA NPs. The NP size was determined to be 177 ± 6 nm by dynamic light scattering (DLS). The encapsulation efficiency of Sim in PLGA was ~94%, as quantified by HPLC. The Sim-PLGA NPs presented the continuous release of Sim, which resulted in 25% of the Sim to release within 20 days at pH 7.0. To test the bioactivity of the Sim-loaded PLGA NPs in changing the macrophage polarization and stimulating browning in vitro, adipogenic differentiation of 3T3-L1 cells was induced in the presence of Sim and Sim-PLGA-NPs. After 10 days of differentiation, we examined the browning markers by flow cytometry assay. Meanwhile, the M2 markers were increased significantly with the treatment of the NPs, confirming that the browning was intrinsically induced. Our results from two independent in vitro experiments indicated that Sim administration in PLGA NPs significantly reduced oil drop formation and changed macrophage polarization. On the other hand, differentiated cells treated with PBS or free drug formed oil drops and did not show an oil reduction. Mechanistically, the treatment decreased the production of IFN-ɣ and IL-2 among lymphocytes, compared with free Sim and PBS. These results suggest that our Sim-PLGA NPs-based strategy may have inhibited fat production and can be used as a treatment for obesity. The results indicated that the Sim-PLGA NPs change macrophage phenotypes to M2 and engineer adipocyte differentiation. The results indicated that treating macrophages with Sim-PLGA NPs (20mg/ml after 72h treatment) can significantly reduce the oil accumulated inside the adipocyte cells in vitro as compared to the pure Sim samples with higher concentrations.

Acknowledgements (Optional):