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. 2025 Dec 3;8(2):595-611.
doi: 10.1039/d5na00706b. eCollection 2026 Jan 20.

Targeted delivery of berberine via ROS-sensitive polymersomes enhances its hepatoprotective activity in CCl4-intoxicated mice

Iva Suman  1 , Damir Klepac  1   2 , Martina Vragović  3 , Hrvoje Križan  1 , Eliézer Jäger  3 , Alessandro Jäger  3 , Ewa Pavlova  3 , Martin Hrubý  3 , Robert Domitrović  1
Affiliations

Targeted delivery of berberine via ROS-sensitive polymersomes enhances its hepatoprotective activity in CCl4-intoxicated mice

Iva Suman et al. Nanoscale Adv. .

Abstract

Carbon tetrachloride (CCl4) metabolism results in the production of highly reactive free radicals and consequent liver tissue damage, making CCl4-induced liver injury an ideal model for studying drug delivery systems that respond to reactive oxygen species (ROS). Previously, we demonstrated the hepatoprotective activity of isoquinoline alkaloid berberine (BER) against CCl4-induced hepatotoxicity in mice. In this study, we aimed to investigate the targeted delivery of BER to ROS-rich injury site. For this purpose, ROS-responsive polymersomes (PS), built as amphiphilic block copolymers bearing a boronic ester-based ROS sensor connected to the hydrophobic polymer backbone with embedded BER, were synthesized in our laboratory. PS exhibited a suitable particle size of 117.8 nm, zeta potential of -12.5 mV, and good physical stability. Mice were administered berberine (BER) and polymersome nanoencapsulated berberine (BER-PS) 6 mg kg-1 intraperitoneally, 1 h before CCl4 (10% v/v in olive oil, 2 mL kg-1) and sacrificed 48 h later. Serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were markedly decreased and histopathological changes were significantly reduced by BER-PS compared to BER. The expression of oxidative stress markers (4-hydroxynonenal (4-HNE), hem oxygenase-1 (HO-1), 8-hydroxy-2'-deoxyguanosine (8-OHdG)), apoptosis (caspase-3, caspase-9, TUNEL), autophagy (microtubule-associated protein 1 light chain 3 beta (LC3B)-I/II, p62), and inflammation (tumor necrosis factor-alpha (TNF-α), nuclear factor kappa B (NF-κB)) was also more effectively ameliorated by BER-PS. Mechanistically, both BER and BER-PS decreased the expression of phosphorylated extracellular signal-regulated kinase (ERK)1/2 and phosphorylated AMP-activated protein kinase (AMPK). BER-PS also decreased nuclear factor-kappa B (NF-κB), tumor necrosis factor-alpha (TNF-α), phosphorylated c-Jun N-terminal kinase (JNK)1/2 and phosphorylated protein kinase B (Akt). These results suggest that BER-PS is more successful than BER in ameliorating ROS-mediated CCl4-induced hepatic injury, which could be related to the specifically targeted delivery of the drug to the site of injury under oxidative stress conditions.

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Conflict of interest statement

There are no conflicts to declare.

Figures

Fig. 1
Fig. 1. Distribution of sizes for BER-loaded PS.
Fig. 2
Fig. 2. Cryo-TEM image of BER-loaded polymersomes (BER-PS); blue arrow shows polymeric bilayer, and red arrow indicates an aqueous core (A). Corresponding size distribution histogram calculated using ImageJ software (B).
Fig. 3
Fig. 3. Release of berberine from BER-loaded PS in PBS and PBS containing 1 mM H2O2.
Fig. 4
Fig. 4. Body weight change and serum markers of liver injury. Mice were treated with vehicle (A), polimersomes (PS) (B), carbon tetrachloride (CCl4) (C), CCl4 + berberine (BER) (D), and CCl4 + BER-PS (E). Each value represents the mean ± SD for 6 mice. ** p < 0.01, *** p < 0.001 CCl4 compared to control; #p < 0.05 CCl4 compared to BER + CCl4 and BER-PS + CCl4; $p < 0.05, $$p < 0.01, $$$p < 0.001 BER + CCl4 compared to BER-PS + CCl4.
Fig. 5
Fig. 5. Hematoxylin and eosin-stained microphotographs of liver tissue. Mice were treated with vehicle (A), polimersomes (PS) (B), carbon tetrachloride (CCl4) (C), CCl4 + berberine (BER) (D), and CCl4 + BER-PS (E). Original magnification × 100. Letter “N” denotes necrotic areas, arrows show microsteatosis. Liver injury score (F). Each value represents the mean ± SD for 6 mice. Representative images from at least 10 random fields (×100). ###p < 0.001 CCl4 compared to BER + CCl4 and BER-PS + CCl4; $$p < 0.01 BER + CCl4 compared to BER-PS + CCl4.
Fig. 6
Fig. 6. Western blot analysis of oxidative stress, apoptosis, and autophagy markers in the liver (A). Mice were treated with vehicle, polimersomes (PS), carbon tetrachloride (CCl4), CCl4 + berberine (BER), and CCl4 + BER-PS. Semiquantification of the expression of 4-hydroxynonenal (4-HNE) (B), hem oxygenase-1 (HO-1) (C), cleaved caspase-3 (D), caspase-9 (E), microtubule-associated protein 1 light chain 3 beta (LC3B)-I/II (F), p62 (G), and p21 (H). Each value represents the mean ± SD for 6 mice. *** p < 0.001 CCl4 compared to control; #p < 0.05, ##p < 0.01, ###p < 0.001 CCl4 compared to BER + CCl4 and BER-PS + CCl4; $p < 0.05, $$p < 0.01, $$$p < 0.001 BER + CCl4 compared to BER-PS + CCl4.
Fig. 7
Fig. 7. Immunofluorescence analysis of 8-hydroxy-2′-deoxyguanosine (8-OHdG) in the liver of mice treated with vehicle (A), polimersomes (PS) (B), carbon tetrachloride (CCl4) (C), CCl4 + berberine (BER) (D), and CCl4 + BER-PS (E). Measurement of the intensity of 8-OHdG immunopositive staining (F). Each value represents the mean ± SD for 6 mice. Representative images from at least 10 high-power fields (×400). Insets: enlarged view of 8-OHdG immunopositive cells (arrows). *** p < 0.001 CCl4 compared to control; #p < 0.05, ##p < 0.01 CCl4 compared to BER + CCl4 and BER-PS + CCl4; $p < 0.05 BER + CCl4 compared to BER-PS + CCl4.
Fig. 8
Fig. 8. TUNEL assay of the liver of mice treated with vehicle (A), polimersomes (PS) (B), carbon tetrachloride (CCl4) (C), CCl4 + berberine (BER) (D), and CCl4 + BER-PS (E). Number of TUNEL-positive hepatocytes (F). Each value represents the mean ± SD for 6 mice. Representative images from at least 10 high-power fields (×400). Insets: enlarged view of TUNEL-positive cells (arrows). #p < 0.05, ###p < 0.001 CCl4 compared to BER + CCl4 and BER-PS + CCl4; $$p < 0.01 BER + CCl4 compared to BER-PS + CCl4.
Fig. 9
Fig. 9. Western blot analysis of mitogen-activated protein kinases and Akt expression in the liver (A). Mice were treated with vehicle, polimersomes (PS), carbon tetrachloride (CCl4), CCl4 + berberine (BER), and CCl4 + BER-PS. Semiquantification of the expression of extracellular signal-regulated kinase (ERK1/2) and activated ERK1/2 (p-ERK1/2) (B), p38 and activated p38 (p-p38) (C), c-Jun N-terminal kinase (JNK1/2) and activated JNK1/2 (p-JNK1/2) (D), and Akt and activated Akt (p-Akt) (E). Each value represents the mean ± SD for 6 mice. * p < 0.05, ** p < 0.01, *** p < 0.001 CCl4 compared to control; #p < 0.05, ##p < 0.01 CCl4 compared to BER + CCl4 and BER-PS + CCl4; $p < 0.05, $$$p < 0.001 BER + CCl4 compared to BER-PS + CCl4.
Fig. 10
Fig. 10. Immunohistochemical staining for nuclear factor kappaB (NF-κB) in the liver. Mice were treated with vehicle (A), polimersomes (PS) (B), carbon tetrachloride (CCl4) (C), CCl4 + berberine (BER) (D), and CCl4 + BER-PS (E). Measurement of NF-κB immunopositive staining intensity (F). Each value represents the mean ± SD for 6 mice. Representative images from at least 10 random fields (×100). Insets: enlarged view of NF-κB-immunopositive cells (arrows). *** p < 0.001 CCl4 compared to control; #p < 0.05, ##p < 0.01, ###p < 0.001 CCl4 compared to BER + CCl4 and BER-PS + CCl4; $p < 0.05 BER + CCl4 compared to BER-PS + CCl4.
Fig. 11
Fig. 11. Immunohistochemical staining for tumor necrosis factor-alpha (TNF-α) in the liver. Mice were treated with vehicle (A), polimersomes (PS) (B), carbon tetrachloride (CCl4) (C), CCl4 + berberine (BER) (D), and CCl4 + BER-PS (E). Measurement of TNF-α immunopositive staining intensity (F). Each value represents the mean ± SD for 6 mice. Representative images from at least 10 random fields (×100). Insets: enlarged view of TNF-α-immunopositive cells (arrows). *** p < 0.001 CCl4 compared to control; #p < 0.05, ###p < 0.001 CCl4 compared to BER + CCl4 and BER-PS + CCl4; $$p < 0.01 BER + CCl4 compared to BER-PS + CCl4.
Fig. 12
Fig. 12. Release of berberine (BER) from berberine-polimersomes (BER-PS) in reactive oxygen species (ROS)-rich environment in the liver due to carbon tetrachloride (CCl4) metabolism, and the mechanism of BER-PS hepatoprotective activity against CCl4 liver injury.
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