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.

Centre for Micro and Nanosciences and Technologies University of Rijeka Radmile Matejčić 2 51000 Rijeka Croatia

Department of Medical Chemistry Biochemistry and Clinical Chemistry Faculty of Medicine University of Rijeka Braće Branchetta 20 51000 Rijeka Croatia

Institute of Macromolecular Chemistry v v i Academy of Sciences of the Czech Republic Heyrovsky Sq 2 162 06 Prague 6 Czech Republic

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Bissell M. D. Gores G. J. Laskin D. L. Hoofnagle J. H. Drug-Induced Liver Injury: Mechanisms and Test Systems. Hepatology. 2001;33(4):1009–1013. PubMed

Unsal V. Cicek M. Sabancilar I. Toxicity of carbon tetrachloride, free radicals and role of antioxidants. Rev. Environ. Health. 2021;36(2):279–295. PubMed

Domitrović R. Jakovac H. Blagojević G. Hepatoprotective activity of berberine is mediated by inhibition of TNF-α, COX-2, and iNOS expression in CCl4-intoxicated mice. Toxicology. 2011;280(1–2):33–43. doi: 10.1016/j.tox.2010.11.005. PubMed DOI

Neag M. A. Mocan A. Echeverría J. Pop R. M. Bocsan C. I. Crişan G. et al., Berberine: Botanical Occurrence, Traditional Uses, Extraction Methods, and Relevance in Cardiovascular, Metabolic, Hepatic, and Renal Disorders. Front. Pharmacol. 2018;9:557. PubMed PMC

Fatima H. S. Saleem H. Khurshid U. Khursheed A. Tauquir Alam M. Imran M. et al., Genus Berberis : A Comprehensive and Updated Review on Ethnobotanical Uses, Phytochemistry and Pharmacological Activities. Chem. Biodiversity. 2024;21(10):e202400911. PubMed

Domitrović R. Potočnjak I. A comprehensive overview of hepatoprotective natural compounds: mechanism of action and clinical perspectives. Arch. Toxicol. 2016;90(1):39–79. PubMed

Gendy A. M. Elnagar M. R. Allam M. M. Mousa M. R. Khodir A. E. El-Haddad A. E. et al., Berberine-loaded nanostructured lipid carriers mitigate warm hepatic ischemia/reperfusion-induced lesion through modulation of HMGB1/TLR4/NF-κB signaling and autophagy. Biomed. Pharmacother. 2022;145:112122. doi: 10.1016/j.biopha.2021.112122. PubMed DOI

Sun Y. Miao D. Zeng Z. Li X. Pu Y. Liu L. et al., Recent Advances in Micro/Nanoneedle Arrays Mediated Intracellular Delivery of Biomacromolecules In Vitro and In Vivo. Adv. Funct. Mater. 2025:2422234.

Jäger E. Sincari V. Albuquerque L. J. C. Jäger A. Humajova J. Kucka J. et al., Reactive Oxygen Species (ROS)-Responsive Polymersomes with Site-Specific Chemotherapeutic Delivery into Tumors via Spacer Design Chemistry. Biomacromolecules. 2020;21(4):1437–1449. doi: 10.1021/acs.biomac.9b01748. PubMed DOI

Singh K. Singhal S. Pahwa S. Sethi V. A. Sharma S. Singh P. et al., Nanomedicine and drug delivery: a comprehensive review of applications and challenges. Nano-Struct. Nano-Objects. 2024;40:101403. doi: 10.1016/j.nanoso.2024.101403. DOI

Miao D. Song Y. De Munter S. Xiao H. Vandekerckhove B. De Smedt S. C. Huang C. Braeckmans K. Xiong R. Photothermal nanofiber-mediated photoporation for gentle and efficient intracellular delivery of macromolecules. Nat. Protoc. 2025;20(7):1810–1845. doi: 10.1038/s41596-024-01115-7. PubMed DOI

Liu L. Hou K. Lin S. Di Y. Zhuang Z. Zeng Z. et al., Hemoglobin based oxygen carrier and its application in biomedicine. Coord. Chem. Rev. 2025;532:216508. doi: 10.1016/j.ccr.2025.216508. DOI

Yun Y. An J. Kim H. J. Choi H. K. Cho H. Y. Recent advances in functional lipid-based nanomedicines as drug carriers for organ-specific delivery. Nanoscale. 2025;17(13):7617–7638. PubMed

Gupta B. Sharma P. K. Malviya R. Carbon Nanotubes for Targeted Therapy: Safety, Efficacy, Feasibility and Regulatory Aspects. Curr. Pharm. Des. 2024;30(2):81–99. doi: 10.2174/0113816128282085231226065407. PubMed DOI

Rajabi M. Feyzbakhsh H. Polymeric nanocarriers in drug delivery, bioimaging, and biosensing: advances and perspectives on nanomicelles, nanogels, and dendrimers. RSC Adv. 2025;15(48):40400–40438. PubMed PMC

Rideau E. Dimova R. Schwille P. Wurm F. R. Landfester K. Liposomes and polymersomes: a comparative review towards cell mimicking. Chem. Soc. Rev. 2018;47:8572–8610. doi: 10.1039/C8CS00162F. PubMed DOI

Kim S. Kim Y. K. Kim S. Choi Y. S. Lee I. Joo H. et al., Dual-mode action of scalable, high-quality engineered stem cell-derived SIRPα-extracellular vesicles for treating acute liver failure. Nat. Commun. 2025;16(1):1903. doi: 10.1038/s41467-025-57133-w. PubMed DOI PMC

Qiu C. Zhang W. Zhao Y. Han T. Yang W. Liu Y. et al., Reprogramming Glucose Metabolism of Macrophage for Acute Liver Failure Therapy with Itaconate Lipo-Nanodrug. Adv. Healthcare Mater. 2025;14(15):e2500019. doi: 10.1002/adhm.202500019. PubMed DOI

Ding Y. Zhang S. Sun Z. Tong Z. Ge Y. Zhou L. et al., Preclinical validation of silibinin/albumin nanoparticles as an applicable system against acute liver injury. Acta Biomater. 2022;146:385–395. PubMed

Shan X. Li J. Liu J. Feng B. Zhang T. Liu Q. et al., Targeting ferroptosis by poly(acrylic) acid coated Mn3O4 nanoparticles alleviates acute liver injury. Nat. Commun. 2023;14(1):7598. doi: 10.1038/s41467-023-43308-w. PubMed DOI PMC

Xia F. Hu X. Zhang B. Wang X. Guan Y. Lin P. et al., Ultrasmall Ruthenium Nanoparticles with Boosted Antioxidant Activity Upregulate Regulatory T Cells for Highly Efficient Liver Injury Therapy. Small. 2022;18(29):e2201558. doi: 10.1002/smll.202201558. PubMed DOI

Fonseca M. Jarak I. Victor F. Domingues C. Veiga F. Figueiras A. Polymersomes as the Next Attractive Generation of Drug Delivery Systems: Definition, Synthesis and Applications. Materials. 2024;17(2):319. PubMed PMC

D'Autréaux B. Toledano M. B. ROS as signalling molecules: mechanisms that generate specificity in ROS homeostasis. Nat. Rev. Mol. Cell Biol. 2007;8(10):813–824. doi: 10.1038/nrm2256. PubMed DOI

Marguet M. Bonduelle C. Lecommandoux S. Multicompartmentalized polymeric systems: towards biomimetic cellular structure and function. Chem. Soc. Rev. 2013;42(2):512–529. doi: 10.1039/C2CS35312A. PubMed DOI

Zuo F. Nakamura N. Akao T. Hattori M. Pharmacokinetics of Berberine and Its Main Metabolites in Conventional and Pseudo Germ-Free Rats Determined by Liquid Chromatography/Ion Trap Mass Spectrometry. Drug Metab. Dispos. 2006;34(12):2064–2072. doi: 10.1124/dmd.106.011361. PubMed DOI

Sawant S. Dongre H. Singh A. K. Joshi S. Costea D. E. Mahadik S. et al., Establishment of 3D Co-Culture Models from Different Stages of Human Tongue Tumorigenesis: Utility in Understanding Neoplastic Progression. PLoS ONE. 2016;11(8):e0160615. doi: 10.1371/journal.pone.0160615. PubMed DOI PMC

Domitrović R. Škoda M. Vasiljev Marchesi V. Cvijanović O. Pernjak Pugel E. Štefan M. B. Rosmarinic acid ameliorates acute liver damage and fibrogenesis in carbon tetrachloride-intoxicated mice. Food Chem. Toxicol. 2013;51:370–378. doi: 10.1016/j.fct.2012.10.021. PubMed DOI

Albuquerque L. J. C. Sincari V. Jäger A. Konefał R. Pánek J. Černoch P. Microfluidic-Assisted Engineering of Quasi-Monodisperse pH-Responsive Polymersomes toward Advanced Platforms for the Intracellular Delivery of Hydrophilic Therapeutics. Langmuir. 2019;35(25):8363–8372. PubMed

De Oliveira F. A. Albuquerque L. J. C. Riske K. A. Jäger E. Giacomelli F. C. Outstanding protein-repellent feature of soft nanoparticles based on poly(N-(2-hydroxypropyl) methacrylamide) outer shells. J. Colloid Interface Sci. 2020;574:260–271. doi: 10.1016/j.jcis.2020.04.048. PubMed DOI

De Oliveira F. A. Batista C. C. D. S. Černoch P. Sincari V. Jäger A. Jäger E. et al., Role of Membrane Features on the Permeability Behavior of Polymersomes and the Potential Impacts on Drug Encapsulation and Release. Biomacromolecules. 2023;24(5):2291–2300. doi: 10.1021/acs.biomac.3c00162. PubMed DOI

De Gracia L. C. Joshi-Barr S. Nguyen T. Mahmoud E. Schopf E. Fomina N. et al., Biocompatible Polymeric Nanoparticles Degrade and Release Cargo in Response to Biologically Relevant Levels of Hydrogen Peroxide. J. Am. Chem. Soc. 2012;134(38):15758–15764. doi: 10.1021/ja303372u. PubMed DOI PMC

Höcherl A. Jäger E. Jäger A. Hrubý M. Konefał R. Janoušková O. et al., One-pot synthesis of reactive oxygen species (ROS)-self-immolative polyoxalate prodrug nanoparticles for hormone dependent cancer therapy with minimized side effects. Polym. Chem. 2017;8(13):1999–2004. doi: 10.1039/C7PY00270J. DOI

Dai C. Xiao X. Li D. Tun S. Wang Y. Velkov T. et al., Chloroquine ameliorates carbon tetrachloride-induced acute liver injury in mice via the concomitant inhibition of inflammation and induction of apoptosis. Cell Death Dis. 2018;9(12):1164. doi: 10.1038/s41419-018-1136-2. PubMed DOI PMC

Morton D. Griffiths P. Guidelines on the recognition of pain, distress and discomfort in experimental animals and an hypothesis for assessment. Vet. Rec. 1985;116(16):431–436. PubMed

Li L. Lan Y. Wang F. Gao T. Linarin Protects Against CCl4-Induced Acute Liver Injury via Activating Autophagy and Inhibiting the Inflammatory Response: Involving the TLR4/MAPK/Nrf2 Pathway. Drug Des. Dev. Ther. 2023;17:3589–3604. doi: 10.2147/DDDT.S433591. PubMed DOI PMC

Han Z. Batudeligen Chen H. Narisu Anda Xu Y. et al., Luteolin attenuates CCl4-induced hepatic injury by inhibiting ferroptosis via SLC7A11. BMC Complement. Med. Ther. 2024;24(1):193. doi: 10.1186/s12906-024-04486-2. PubMed DOI PMC

Peng X. Dai C. Liu Q. Li J. Qiu J. Curcumin Attenuates on Carbon Tetrachloride-Induced Acute Liver Injury in Mice via Modulation of the Nrf2/HO-1 and TGF-β1/Smad3 Pathway. Molecules. 2018;23(1):215. doi: 10.3390/molecules23010215. PubMed DOI PMC

Liu Y. Wen P. Zhang X. Dai Y. He Q. Breviscapine ameliorates CCl4-induced liver injury in mice through inhibiting inflammatory apoptotic response and ROS generation. Int. J. Mol. Med. 2018;42(2):755–768. PubMed PMC

Nössing C. Ryan K. M. 50 years on and still very much alive: ‘Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br. J. Cancer. 2023;128(3):426–431. PubMed PMC

Han C. Sun T. Xv G. Wang S. Gu J. Liu C. Berberine ameliorates CCl4-induced liver injury in rats through regulation of the Nrf2-Keap1-ARE and p53 signaling pathways. Mol. Med. Rep. 2019;20(4):3095–3102. PubMed PMC

Hwang H. J. Ha H. Lee B. S. Kim B. H. Song H. K. Kim Y. K. LC3B is an RNA-binding protein to trigger rapid mRNA degradation during autophagy. Nat. Commun. 2022;13(1):1436. doi: 10.1038/s41467-022-29139-1. PubMed DOI PMC

Liu W. J. Ye L. Huang W. F. Guo L. J. Xu Z. G. Wu H. L. et al., p62 links the autophagy pathway and the ubiqutin–proteasome system upon ubiquitinated protein degradation. Cell. Mol. Biol. Lett. 2016;21(1):29. doi: 10.1186/s11658-016-0031-z. PubMed DOI PMC

Zhuang W. Huang Z. Yu L. Yu M. He H. Deng Y. Berberine enhances autophagic flux to alleviate ischemic neuronal injury by facilitating N-ethylmaleimide-sensitive factor-mediated fusion of autophagosomes with lysosomes. Biochem. Pharmacol. 2025;232:116715. doi: 10.1016/j.bcp.2024.116715. PubMed DOI

Xu X. Li W. Yu Z. Zhang L. Duo T. Zhao Y. et al., Berberine Ameliorates Dextran Sulfate Sodium-Induced Ulcerative Colitis and Inhibits the Secretion of Gut Lysozyme via Promoting Autophagy. Metabolites. 2022;12(8):676. doi: 10.3390/metabo12080676. PubMed DOI PMC

Hu F. Hu T. Qiao Y. Huang H. Zhang Z. Huang W. et al., Berberine inhibits excessive autophagy and protects myocardium against ischemia/reperfusion injury via the RhoE/AMPK pathway. Int. J. Mol. Med. 2024;53(5):49. doi: 10.3892/ijmm.2024.5373. PubMed DOI PMC

Jung D. Khurana A. Roy D. Kalogera E. Bakkum-Gamez J. Chien J. et al., Quinacrine upregulates p21/p27 independent of p53 through autophagy-mediated downregulation of p62-Skp2 axis in ovarian cancer. Sci. Rep. 2018;8(1):2487. doi: 10.1038/s41598-018-20531-w. PubMed DOI PMC

Singh A. Handa M. Ruwali M. Flora S. J. S. Shukla R. Kesharwani P. Nanocarrier mediated autophagy: An emerging trend for cancer therapy. Process Biochem. 2021;109:198–206. doi: 10.1016/j.procbio.2021.07.011. DOI

Kma L. Baruah T. J. The interplay of ROS and the PI3K/Akt pathway in autophagy regulation. Biotechnol. Appl. Biochem. 2022;69(1):248–264. doi: 10.1002/bab.2104. PubMed DOI

Bak J., Je N. K., Chung H. Y., Yokozawa T., Yoon S. and Moon J. O.. Oligonol Ameliorates CCl4 -Induced Liver Injury in Rats PubMed PMC

Feng X. W. Cheng Q. L. Fang L. Liu W. Y. Liu L. W. Sun C. Q. et al., Corn oligopeptides inhibit Akt/NF-κB signaling pathway and inflammatory factors to ameliorate CCl4 -induced hepatic fibrosis in mice. J. Food Biochem. 2022;46(8):e14162. doi: 10.1111/jfbc.14162. PubMed DOI

Liao C. C. Day Y. J. Lee H. C. Liou J. T. Chou A. H. Liu F. C. ERK Signaling Pathway Plays a Key Role in Baicalin Protection Against Acetaminophen-Induced Liver Injury. Am. J. Chin. Med. 2017;45(01):105–121. doi: 10.1142/S0192415X17500082. PubMed DOI

Yue J. López J. M. Understanding MAPK Signaling Pathways in Apoptosis. Int. J. Mol. Sci. 2020;21(7):2346. doi: 10.3390/ijms21072346. PubMed DOI PMC

Miura H. Kondo Y. Matsuda M. Aoki K. Cell-to-Cell Heterogeneity in p38-Mediated Cross-Inhibition of JNK Causes Stochastic Cell Death. Cell Rep. 2018;24(10):2658–2668. doi: 10.1016/j.celrep.2018.08.020. PubMed DOI

Polyzos S. A. Kountouras J. Zavos C. Nonalcoholic fatty liver disease: the pathogenetic roles of insulin resistance and adipocytokines. Curr. Mol. Med. 2009;9(3):299–314. doi: 10.2174/156652409787847191. PubMed DOI

Tiegs G. Horst A. K. TNF in the liver: targeting a central player in inflammation. Semin. Immunopathol. 2022;44(4):445–459. doi: 10.1007/s00281-022-00910-2. PubMed DOI PMC