Nanodrug with ROS and pH Dual‐Sensitivity Ameliorates Liver Fibrosis via Multicellular Regulation

Abstract Liver fibrosis currently represents a global health problem without effective pharmacotherapeutic strategies. The clinical translation of polydatin, a promising natural anti‐fibrotic drug candidate with broad anti‐inflammatory and antioxidant capabilities, remains a major challenge due to its limited water solubility and tissue absorption. Herein, a polydatin‐loaded micelle (PD‐MC) based on reactive oxygen species (ROS) and pH dual‐sensitive block polymer PEG‐P(PBEM‐co‐DPA) is developed. The micelle exerts great potential in improving the biocompatibility of polydatin and shows highly efficient liver‐targeted drug release in response to the fibrotic microenvironment. Both in vitro and in vivo studies demonstrate that PD‐MC can significantly suppress inflammatory response and oxidative stress, reduce hepatocyte apoptosis, and avert activation of macrophages and hepatic stellate cells. More excitingly, the blank micelle itself promotes the hepatic ROS consumption at the pathologic site to provide anti‐inflammatory benefits. These favorable therapeutic virtues of targeting multiple cell types endow PD‐MC with remarkable efficacy with minimal side effects in liver fibrosis treatment. Thus, PD‐MC holds great potential to push forward the clinical application of polydatin in pharmacotherapeutic approaches against liver fibrosis.

The reaction were kept stirring at room temperature for 12 h under nitrogen protection.
Then the mixture was filtered, concentrated, re-dissolved in ethyl acetate. The solution was precipitated into cold excessive diethyl ether and washed with diethyl ether twice. Finally, the precipitated was vacuum-dried to obtain the product 4-(hydroxymethyl)phenylboronic acid pinacol ester (6.3 g, yield: 68.4%).

Synthesis of ROS-responsive monomer
The tissues of major organs (heart, liver, spleen, lung and kidney) were fixed in 10% neutral buffered formalin, embedded in paraffin and made into 4 μm sections. Then the liver sections were deparaffinized, hydrated and stained following standard methods. For immunochemistry staining, liver sections were deparaffinized, rehydrated, and incubated with 3% hydrogen peroxide in order to block endogenous peroxidase. After heated in 10 mM sodium citrate buffer to retrieve antigen and blocked by 5% BSA solution, the liver sections were sequentially incubated with primary antibodies (CD68, TLR4 and α-SMA) and secondary antibodies. After counterstained with hematoxylin, the liver sections were mounted with neutral balsam medium. For immunofluorescent staining, the liver tissues were fixed in 10% PBS-buffered formalin overnight, sequentially exposed to 10% and 30% sucrose in PBS for 10 h each and finally embedded in Tissue Tek OTC compound (Sakura Finetek, Torrance, CA). After permeabilized by 0.25% Triton X-100, the liver sections were sequentially incubated with primary antibodies (Cleaved-caspase3, NF-κB p-p65, 4-HNE and NOX-4) and corresponding Alexa Fluor 488 or 594-conjugated secondary antibodies. Additionally, the in vitro immunofluorescent analyses for RAW (NF-κB p65) and LX-2 (α-SMA) cells were performed in a similar way as described above. Finally, the areas of interest were captured by an Olympus BX51 microscope (Olympus Co., Tokyo, Japan).
Liver and kidney functional assay: Serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (Cr) and urea were measured using standard enzymatic procedures following the manufacturer's instruction.
Hydroxyproline assay: Hepatic hydroxyproline content was assessed using commercial hydroxyproline assay kit following the manufacturer's instructions.
Briefly, the liver tissues were hydrolyzed at 95 ℃ for 25 min, adjusted to pH 6.5 and then filtered through activated charcoal. After centrifugation, the supernatant was collected, mixed with detecting liquid and incubated at 60 ℃ for 20 min. Finally, the samples were measured by a microplate reader at 550 nm.

Evaluation of Nitric oxide (NO) availability:
The liver sinusoidal endothelial cells (LSECs) were isolated from the C57BL/6 mice induced by CCl 4 for 6 weeks according to the method of a previous report [3] . The LSECs isolated from fibrotic mice were treated as follows: (1)  Measurement of TXB 2 : The LSECs isolated from fibrotic mice were treated as described above. After 24 h, arachidonic acid (40 μM) was added for 30 min in order to stimulate the prostanoid production of LSECs. Next, the supernatant was collected and the TXA 2 (using its stable metabolites TXB 2 ) was analyzed by an enzyme-linked immunosorbent assay kit (Cayman Chemical, Ann Arbor, MI, USA) according to the manufacturer's instructions. For the in vivo study, a flow-controlled perfusion system was conducted in fibrotic mice as previously described [4] . The perfusate was collected and quantified for the analysis of TXB 2 .