Non-alcoholic fatty liver disease (NAFLD) is one of the most prevalent liver diseases, affecting about 25% of the world's population [1]. The pathogenesis of NAFLD is complex and diverse including gene mutation, environmental factors, and metabolism abnormity. With the excess store of fat in the liver, the pathological changes experience a successive progress from hepatic steatosis to hepatic fibrosis, hepatic cirrhosis, and even hepatic cancer [2,3]. Regrettably, there isn't any FDA-approved specific drug for NAFLD treatment at present. Thus exploring new and effective therapeutic strategies is urgently needed.

Recently, several biomolecules have attracted widespread attention as therapeutic targets owning the potentials to relieve NAFLD [4]. Sirtuin1 (SIRT1), an NAD+ dependent histone/protein deacetylase, is involved in the developmental regulation of liver steatosis, oxidative stress, and insulin resistance. Specific deletion of SIRT1 would result in hepatic steatosis, inflammation and endoplasmic reticulum stress [5]. Besides, nuclear receptor superfamily member FXR is a bile acid receptor that primarily expresses in the liver and controls downstream gene expression to modulate fat synthesis, cholesterol balance and oxidative stress [6], [7], [8]. Activating FXR can improve insulin sensitivity of liver and reduce steatosis by inhibiting fat production. In animal models, FXR agonists can attenuate atherosclerosis and exert indirect anti-fibrotic effects [9]. These characteristics make FXR an attractive therapeutic target for NAFLD. MiR-34a is overexpressed in hepatocytes of HFD-induced or inherited obese mice, as well as NAFLD patients [10,11]. One study previously validated that miR-34a is a major regulator of the occurrence and progress of various metabolic diseases by coordinating regulation of lipid synthesis and transport, inflammatory reaction, ROS generation and apoptosis in hepatocytes [12].

To be noticed, some complicated interactions occur among FXR, miR-34a and SIRT1. Firstly, when FXR is activated, small heterodimer partner (SHP) is recruited to the miR-34a promoter, and suppresses the transcription of miR-34a by competitively inhibiting p53 which is a primary activator of the miR-34a gene [13]. Then, inhibiting miR-34a could increase SIRT1 expression because miR-34a could bind to the 3′ untranscribed region (3′ UTR) of SIRT1 mRNA and block the translation of SIRT1 protein [14], [15], [16]. In addition, SIRT1 can restrain the expression of miR-34a by deacetylation of p53 at the miR-34a promoter and promote the trans-activation potential of FXR by deacetylation of FXR [17,18]. This implies a potential cascade loop among FXR, miR-34a and SIRT1 for NAFLD therapy.

Based on the above information, we propose a therapeutic strategy that simultaneously activates FXR and silences miR-34a, and subsequently increases the expression level of SIRT1, thus consequently treat NAFLD via FXR/miR-34a/SIRT1 loop [19]. Obeticholic acid (OCA), a semi-synthetic bile acid, is a strong and highly selective FXR agonist. Some research has demonstrated that OCA can alleviate insulin resistance and reduce triglyceride levels in the liver in mouse models of hereditary or diet-related obesity. It is considered that OCA can alleviate the deposition of hepatic triglycerides by activating FXR and down-regulating the expression of adipogenesis related genes [20]. On the other hand, miR-34a antagomir (anta-miR-34a) would certainly block the action of miR-34a. However, how to co-deliver OCA and anta-miR-34a would be a predominant issue to in vivo therapeutic effect due to their entirely different physicochemical properties and action mechanisms.

Recently, nanoparticles based on multitudinous biomaterials have been validated as promising medication technology to enhance the treatment effect of drug against NAFLD and other diseases [21], [22], [23], [24]. This study attempted to deliver OCA and anta-miR-34a synchronously to treat NAFLD via a nano-scaled vesical carrier. We designed and synthesized an amphiphilic oligochitosan derivative named UBC, which can self-assemble to nanovesicles containing aqueous center cavity and hydrophobic membrane for the co-loading of hydrophilic anta-miR-34a and hydrophobic OCA, respectively (Scheme 1). Moreover, due to esterase responsive hydrolysis of UBC, the release of OCA and anta-miR-34a could be triggered in the presence of esterase after the nanovesicles undergo cellular internalization. Afterwards, OCA and anta-miR-34a would exert synergetic action through FXR/miR-34a/SIRT1 loop pathway to ameliorate NAFLD. To validate our assumption, the comprehensive investigations to characterize the drug-loaded nanovesicles were carried out. In addition, we demonstrated the presence of a regulatory loop by detecting the expression levels of target proteins and genes and observing the role of UBC/OCA/anta-miR-34a in reducing lipid deposition in high-fat cells and HFD-induced mouse models.