Microenvironment Activatable Nanoprodrug Based on Gripper-like Cyclic Phenylboronic Acid to Precisely and Effectively Alleviate Drug-induced Hepatitis


Abstract

Drug-induced hepatitis (DIH), which seriously interferes with disease treatment, is one of the most common reasons for termination of new drugs during preclinical studies or post-marketing surveillance. Although antioxidants and anti-inflammatory agents are promising, their nonspecific distribution and insolubility limit their application. Therefore, precise drug release at the disease site is an important way to alleviate DIH and avoid side effects. Methods: A gripper-like hydrophilic cyclic phenylboronic acid (cPBA) was synthesized and a nanoprodrug (cPBA-BE) was established by coupling cPBA with hydrophobic baicalein (BE). The stimuli-responsive release properties and therapeutic effect of cPBA-BE on drug-injured hepatocyte were investigated. The biodistribution and therapeutic effect of cPBA-BE both in acetaminophen-induced acute hepatitis model and rifampicin-induced chronic hepatitis model were further evaluated. Results: cPBA-BE conjugate could self-assemble into nanoprodrug with cPBA as the hydrophilic external layer and BE as the hydrophobic core. In HepaRG cells, cPBA-BE showed stronger cellular uptake. Due to the H2O2- and acid-sensitivity, cPBA-BE could achieve adequate BE release, significantly resist the depletion of GSH, mitochondrial dysfunction, downregulation of inflammation and cell apoptosis in the acetaminophen injured HepaRG cells. Biodistribution showed that cPBA-BE specifically increased the concentration of BE in the liver of DIH mice. cPBA-BE could alleviate acetaminophen-induced acute hepatitis or rifampicin-induced chronic hepatitis more effectively through relieving the oxidative stress, inflammation and block the neutrophil infiltration in liver. Conclusions: cPBA is expected to be a good platform for constructing injectable nanoprodrug with both H2O2 and pH-responsive properties by coupling a wide range of drugs containing o-diol. In this study, the nanoprodrug cPBA-BE was determined to be effective for alleviating the DIH.

Keywords: Inflammation; Injectable-nanoprodrug; Microenvironment-activatable; Self-assembly; Targeted therapy.

Conflict of interest statement

Competing Interests: Q.X.Z., S.S.L., K.G. and R.S.T. have filed a patent application relating to the content of this work. All other authors have no competing interest to declare.

Figures

Scheme 1
Scheme 1
Schematic illustration for the preparation and application of the cPBA-BE nanoprodrug. Baicalein (BE) is coupled to the gripper-like circular PBA (cPBA) via the reaction between o-diol and PBA to form cPBA-BE amphiphilic monomer, then nanoprodrug cPBA-BE is formed by self-assembly (A). Passive and precise delivery of BE to the liver tissue of hepatitis. The nanoprodrug cPBA-BE is disassembled by excessive ROS and acidic environment in DIH to release the BE (B). HMP refers to 4-hydroxymethyl-phenol.
Figure 1
Figure 1
Synthesis and characterization of cPBA. A, The synthetic route of cPBA. B-C, The FT-IR (B) and (C) 1H NMR spectra in D2O of cPBA.
Figure 2
Figure 2
Interaction between cPBA and BE. A, Fluorescence quenching of cPBA after an interaction with various concentrations (0, 3, 6, 12, 24 μg/mL) of BE. B, Plotting of BE concentration-dependent steady-state fluorescence quenching of cPBA using the Stern-Volmer equation at different pH. I0 and I represent the fluorescence intensity without or with different concentrations of BE, respectively. C, Detecting the lowest concentration of BE for cPBA-BE self-assembly by dynamic light scattering. 2 mg/mL cPBA was in the system.
Figure 3
Figure 3
Characterization of cPBA-BE nanoprodrug. A, TEM images of cPBA-BE in different media. B-D, Size distribution (B), polydispersity (C) and average hydrodynamic diameter (D) of cPBA-BE exposed in various concentrations (0, 0.125, 0.25, 0.5 or 1 mM) of H2O2. E-G, Size distribution (E), polydispersity (F) and average hydrodynamic diameter (G) of cPBA-BE in PBS with various pH (5.6, 6.4, 7.4, 9.0 or 11.0). All data are presented as mean ± SD (n = 6).
Figure 4
Figure 4
Microenvironment activated drug release. A, Sensitivity of cPBA-BE against various ROS. B-C, BE release profiles in PBS with various concentrations of H2O2 (B) or at different pH values (C). D, Effect of glucose on BE release from cPBA-BE. Normal blood glucose concentration is about 6 mmol/L. All data are presented as mean ± SD (n = 6).
Figure 5
Figure 5
The uptake profiles of cPBA-BE in HepaRG cells. A-B, Representative flow cytometry curves (A) and quantitative data (B) illustrating concentration-dependent internalization of Cy5-labled cPBA-BE in HepaRG cells for 2 h. C-D, Representative flow cytometry curves (C) and quantitative data (D) illustrating time-dependent internalization of 10 mg/mL Cy5-labled cPBA-BE in HepaRG cells till 24 h. All data are presented as mean ± SD (n = 6).
Figure 6
Figure 6
In vitro anti-oxidative, anti-inflammatory and anti-apoptosis activity of cPBA-BE in APAP-injured HepaRG cells. HepaRG cells were cultured with 3 mg/mL of APAP, 80 μg/mL cPBA-BE and 20 μg/mL BE were as treatment groups. A, Intracellular drug release of cPBA-BE in HepaRG cells injured by 3 mg/mL APAP. B, The content of GSH in APAP-injured HepaRG cells determined by kits. C-D, Typical flow cytometry profiles (C) and quantitative analysis (D) showing the intracellular generation of ROS in HepaRG cells. DCFH-DA was used as intracellular ROS probe. E, The expression level of TNF-α in APAP-injured HepaRG cells. F-G, Flow cytometric profiles (F) and quantitative analysis (G) illustrating disruption of mitochondrial membrane potential (ΔΨm). H-I, Representative flow cytometry curves (H) and quantitative data (I) of LPO degree in APAP-injured HepaRG cells. J-K, Typical flow cytometry (J) and quantitative data (K) of HepaRG cell apoptosis. All data are presented as mean ± SD (n = 6). *p < 0.05, **p < 0.01 and ***p < 0.001; ns, no significance.
Figure 7
Figure 7
Biodistribution and pharmacokinetics of cPBA-BE in AIH mice. A, Scheme of establishment of AIH mice and treatment regimens. B, Quantitative analysis of BE concentrations in blood. C, Cumulative amount of BE in blood by calculating the area under the curve (AUC). D-E, The BE concentrations (D) and cumulative amount of BE (E) in the liver of AIH mice. F-G, Representative ex vivo images (F) and quantitative analysis of radiation (G) illustrating distribution of Cy7.5 fluorescence signals in liver tissues. In the experiments of Figure B-E, the mice received 20 mg/mL cPBA-BE or 5.1 mg/mL free BE. For Figures F-G, the mice received Cy7.5-labeled cPBA-BE at a dose of 0.5 mg Cy7.5 in each mouse. All data are presented as mean ± SD (n = 3). *p < 0.05, **p < 0.01 and ***p < 0.001; ns, no significance.
Figure 8
Figure 8
In vivo efficacy of cPBA-BE for alleviating APAP-induced acute hepatitis. A, Establishment of AIH mice and treatment regimens. After mice injured by 200 mg/kg APAP for 6 h, single intravenous injection of 100 μL 20 mg/mL cPBA-BE in PBS or 5.1 mg/mL BE in 5% DMSO (equal to 20 mg/mL cPBA-BE) was performed. B-E, The expression levels of representative factors of inflammation and oxidative stress in liver. After 18 h of treatment, homogenates of the hepatic tissues were prepared, and the concentrations of GSH (B), H2O2 (C), MDA (D) and MPO (E) were separately measured. F-G, Representative flow cytometric analysis (F) and quantitative analysis (G) of the proportion of neutrophils in liver tissues. H-K. The AST (H), ALT (I), LDH (J), ammonia (K) levels in serum. All data are presented as mean ± SD (n = 5). *p < 0.05, **p < 0.01 and ***p < 0.001; ns, no significance.
Figure 9
Figure 9
Histological evaluation of APAP-induced acute hepatitis. A, Organ index of liver in AIH mice. B, H&E-stained histological sections of hepatic tissues from AIH mice with or without various treatments at two magnifications (100× and 200×). Black, blue, and green arrows indicate immune cell infiltration, hepatocyte dilation, and necrosis area, respectively. C, Histological scores of hepatitis in mice. D, Masson trichrome (MT) staining of liver sections. E-F, Analysis of hepatic cell apoptosis by TUNEL assay. All scale bar represents 200 μm. All data are presented as mean ± SD. For A, n = 5, for C and F, n = 10. *p < 0.05, **p < 0.01 and ***p < 0.001; ns, no significance.
Figure 10
Figure 10
Therapeutic effect of cPBA-BE on RFP-induced chronic hepatitis. A, Establishment of RIH mice and treatment regimens. After oral administration of RFP for 6 h, single intravenous injection of 100 μL 20 mg/mL cPBA-BE in PBS or 5.1 mg/mL BE in 5% DMSO was performed. This process lasted 12 days. B, The MDA content in liver reflecting the degree of lipid peroxidation. C-E, The AST (C), ALT (D) and LDH (E) levels in plasma. F, Organ index of liver in RIH mice. All data are presented as mean ± SD (n = 5). G, H&E-stained histological sections of hepatic tissues from RIH mice with or without various treatments. The magnification is 100 and 200 times. Black, blue, and green arrows indicate immune cell infiltration, hepatocyte dilation, and necrosis area, respectively. *p < 0.05, **p < 0.01 and ***p < 0.001; ns, no significance.
See this image and copyright information in PMC

Similar articles