Endosomal disruption by co-encapsulating gentamicin in lipid nanoparticles for efficient siRNA delivery and cancer therapy

Abstract

Efficient siRNA delivery is highly desirable for disease treatment. However, the application of conventional nanoparticles is limited by the inability to escape from endo-lysosomes. Herein, we report a strategy using small-molecule drugs to enhance siRNA endo‐lysosomal release,addressing this challenge. We encapsulated gentamicin(GM) into the marketed Onpattro® formulation to establish LNP-siRNA/GM nanoparticles that promote siRNA endo‐lysosomal escape through endosomal disruption, mechanistically exhibiting unique functionality and synergistic effects of LNP-siRNA/GM to improve cancer therapy. Besides, GM induced reactive oxygen species (ROS) and phospholipids accumulation in endo‐lysosomes, as well as the physical characteristics of lipid nanoparticles (LNPs) were preserved. We also revealed that GM causes endo‐lysosomal swelling and disrupts the endosomal membrane to enable siRNA release, as confirmed by Galectin 3 recruitment and acridine orange release. This approach achieved ∼81% mRNA-EGFR silencing, which is more than LNP-siEGFR (∼56.23%) by enhancing siRNA endo‐lysosomal escape efficiency. Meanwhile, LNP-siEGFR/GM exhibited significant biological activities in HepG2 cells, driven by the synergistic effects of siEGFR and GM with the VEGF and CXCL12 downregulation of, and ROS and phospholipids upregulation. Furthermore, tumor growth was notably suppressed after intravenous injection of LNP-siEGFR/GM in tumor-bearing nude mice. The combination of EGFR-siRNA and GM could also greatly inhibit angiogenesis, be antiproliferative, and induce tumor cells apoptosis. Therefore, this GM and siRNA co-delivery system would provide an efficient strategy for siRNA endosomal escape, significantly improving knockdown in various LNPs based siRNA delivery systems and efficiently enhancing cancer therapy.

Keywords

Sirna delivery / Gentamicin / Endosomal disruption / Cancer therapy

Conflicts of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.

Acknowledgements

This work was supported by National Natural Science Foundation of China (81502688), Cooperation Research Funding of Capital Medical University (2020KJ000514), Cooperation Research Funding of Capital Medical University (2023KJ000814), and R&D Program of Beijing Municipal Education Commission (KM202210025024).

Supplementary materials

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.ajps.2024.101011. The figures and tables with "S" before the serial number are included in the Supplementary material.

References

[1] Li Y, Ye Z, Yang H, Xu Q. Tailoring combinatorial lipid nanoparticles for intracellular delivery of nucleic acids, proteins, and drugs. Acta Pharm Sin B 2022; 12:2624-39.

[2] Hedlund H, Du Rietz H, Johansson JM, Eriksson HC, Zedan W, Huang L, et al. Single-cell quantification and dose-response of cytosolic siRNA delivery. Nat. Commun 2023; 14:1075.

[3] Jiang L, Qi Y, Yang L, Miao Y, Ren W, Liu H, et al. Remodeling the tumor immune microenvironment via siRNA therapy for precision cancer treatment. Asian J Pharm Sci 2023;18: 100852.

[4] Uddin N, Binzel DW, Shu D, Fu TM, Guo P. Targeted delivery of RNAi to cancer cells using RNA-ligand displaying exosome. Acta Pharm Sin B 2023; 13:1383-99.

[5] Moazzam M, Zhang M, Hussain A, Yu X, Huang J, Huang Y. The landscape of nanoparticle-based siRNA delivery and therapeutic development. Molecular Ther 2024; 32:284-312.

[6] Chen S, Zhang S, Wang Y, Yang X, Yang H, Cui C. Anti-EpCAM functionalized graphene oxide vector for tumor targeted siRNA delivery and cancer therapy. Asian J Pharm Sci 2021; 16:598-611.

[7] Cao Z, Liu J, Yang X. Deformable nanocarriers for enhanced drug delivery and cancer therapy. Exploration 2024; 4:20230037.

[8] Tzng E, Bayardo N, Yang PC. Current challenges surrounding exosome treatments. Extracellular Vesicle 2023; 2:100023.

[9] Jeong M, Lee Y, Park J, Jung H, Lee H. Lipid nanoparticles (LNPs) for in vivo RNA delivery and their breakthrough technology for future applications. Adv Drug Delivery Rev 2023; 200:114990.

[10] Yan Y, Liu XY, Lu A, Wang XY, Jiang LX, Wang JC. Non-viral vectors for RNA delivery.J Controlled Release 2022; 342:241-79.

[11] Ma Y, Li S, Lin X, Chen Y. A perspective of lipid nanoparticles for RNA delivery. Exploration 2024; 4:20230147.

[12] Chatterjee S, Kon E, Sharma P, Peer D. Endosomal escape: a bottleneck for LNP-mediated therapeutics. Proc Natl Acad Sci 2024; 121:e2307800120.

[13] Mo Y, Cheng MHY, D'Elia A, Doran K, Ding L, Chen J, et al. Light-Activated siRNA Endosomal Release (LASER) by Porphyrin Lipid Nanoparticles. ACS Nano 2023; 17:4688-703.

[14] Gilleron J, Querbes W, Zeigerer A, Borodovsky A, Marsico G, Schubert U, et al. Image-based analysis of lipid nanoparticle-mediated siRNA delivery, intracellular trafficking and endosomal escape. Nat. Biotechnol. 2013; 31:638-46.

[15] Butowska K, Han X, Gong N, El-Mayta R, Haley RM, Xue L, Zhong W, et al. Doxorubicin-conjugated siRNA lipid nanoparticles for combination cancer therapy. Acta Pharm Sin B 2023; 13:1429-37.

[16] Cullis PR, Hope MJ. Lipid nanoparticle systems for enabling gene therapies. Molecular Ther 2017; 25:1467-75.

[17] Liu S, Cheng Q Wei T, Yu X, Johnson LT, Farbiak L, et al. Membrane-destabilizing ionizable phospholipids for organ-selective mRNA delivery and CRISPR-Cas gene editing. Nat Mater 2021; 20:701-10.

[18] Kim IG, Jung WH, You G, Lee H, Shin YJ, Lim SW, et al. Efficient delivery of globotriaosylceramide synthase siRNA using polyhistidine-incorporated lipid nanoparticles. Macromol Biosci 2023; 23:e2200423.

[19] Moghassemi S, Dadashzadeh A, Azevedo RB, Feron O, Amorim CA. Photodynamic cancer therapy using liposomes as an advanced vesicular photosensitizer delivery system. J Controlled Release 2021; 339:75-90.

[20] Van de Vyver T, Bogaert B, De Backer L, Joris F, Guagliardo R, Van Hoeck J, et al. Cationic amphiphilic drugs boost the lysosomalescape of small nucleic acid therapeutics in a nanocarrier-dependent manner. ACS Nano 2020; 14:4774-91.

[21] Allemailem KS, Almatroudi A, Alrumaihi F, Almatroodi SA, Alkurbi MO, Basfar GT, et al. Novel approaches of dysregulating lysosome functions in cancer cells by specific drugs and its nanoformulations: a smart approach of modern therapeutics. Int J Nanomedicine 2021; 16:5065-98.

[22] Yu NX, Wang XY, Qiu L, Cai TM, Jiang CJ, Sun Y, et al. Bacteria-triggered hyaluronan/AgNPs/gentamicin nanocarrier for synergistic bacteria disinfection and wound healing application. Chem Eng J 2020; 380:122582.

[23] Liang Y, Liang Y, Zhang H, Guo B. Antibacterial biomaterials for skin wound dressing. Asian J Pharm Sci 2022; 17:353-84.

[24] Cuccarese MF, Singh A, Amiji M, O'Doherty GA. A novel use of gentamicin in the ROS-mediated sensitization of NCI-H460 lung cancer cells to various anticancer agents. ACS Chem Biol 2013; 8:2771-7.

[25] Denamur S, Tyteca D, Marchand-Brynaert J, Van Bambeke F, Tulkens PM, Courtoy PJ, et al. Role of oxidative stress in lysosomal membrane permeabilization and apoptosis induced by gentamicin, an aminoglycoside antibiotic. Free Radical Biol Med 2011; 51:1656-65.

[26] Hirode M, Ono A, Miyagishima T, Nagao T, Ohno Y, Urushidani T. Gene expression profiling in rat liver treated with compounds inducing phospholipidosis. Toxicol Appl Pharmacol 2008; 229:290-9.

[27] Nioi P, Pardo ID, Snyder RD. Monitoring the accumulation of fluorescently labeled phospholipids in cell cultures provides an accurate screen for drugs that induce phospholipidosis. Drug Chem Toxicol 2008; 31:515-28.

[28] Quiros Y, Vicente-Vicente L, Morales AI, Lopez-Novoa JM, Lopez-Hernandez FJ. An integrative overview on the mechanisms underlying the renal tubular cytotoxicity of gentamicin. Toxicol Sci 2010; 119:245-56.

[29] Stark M, Silva TFD, Levin G, Machuqueiro M, Assaraf YG. The lysosomotropic activity of hydrophobic weak base drugs is mediated via their intercalation into the lysosomal membrane. Cells 2020; 9(5):1082.

[30] Nowacki MP, Rutkowski A, Olędzki J, Chwaliński M. Prospective, randomized trial examining the role of gentamycin-containing collagen sponge in the reduction of postoperative morbidity in rectal cancer patients: early results and surprising outcome at 3-year follow-up. Int J Colorectal Dis 2004; 20:114-20.

[31] Potdar T, Kontorinis G. Pre-habilitation with intratympanic gentamicin in vestibular schwannomas: a systematic review. J Laryngol Otol 2023; 137:985-91.

[32] Li Y, Ding Q, Gao J, Li C, Hou P, Xu J, et al. Novel mechanisms underlying inhibition of inflammation-induced angiogenesis by dexamethasone and gentamicin via PI3K/AKT/NF- $\kappa$ B/VEGF pathways in acute radiation proctitis. Sci Rep 2022;12: 14116.

[33] Olekson MA, Rose LF, Carlsson AH, Fletcher JL, Leung KP, Chan RK. Ultrahigh dose gentamicin alters inflammation and angiogenesis in vivo and in vitro. Wound Repair and Regeneration 2017; 25:632-40.

[34] Yan Y, Wang CH, Cui SH, Zhai L, Sun J, Liu XY, et al. Enhanced cancer therapeutic efficiency of NO combined with siRNA by caspase-3 responsive polymers. J Control Release 2021; 339:506-20.

[35] Yu S, Zhao R, Zhang B, Lai C, Li L, Shen J, et al. Research progress and application of the CRISPR/Cas9 gene-editing technology based on hepatocellular carcinoma. Asian J Pharm Sci 2023; 18:100828.

[36] Liu Y, Ma XX, Zhu YJ, Lv X, Wang PP, Feng LZ. pH-responsive nanomedicine co-encapsulated with Erlotinib and chlorin e6 can enable effective treatment of triple negative breast cancer via reprogramming tumor vasculature. Chem Eng J 2022; 437:135305.

[37] Fang W, Li L, Lin Z, Zhang Y, Jing Z, Li Y, et al. Engineered IL-15/IL-15R a -expressing cellular vesicles promote T cell anti-tumor immunity. Extracellular Vesicle 2023; 2:100021.

[38] Quiros Y, Vicente-Vicente L, Morales AI, López-Novoa JM, FJ López-Hernández. An integrative overview on the mechanisms underlying the renal tubular cytotoxicity of gentamicin. Toxicological Sci 2011; 119:245-56.

[39] Servais H, Jossin Y, Van Bambeke F, Tulkens PM, MP Mingeot-Leclercq. Gentamicin causes apoptosis at low concentrations in renal LLC-PK1 cells subjected to electroporation. Antimicrob. Agents Chemother. 2006; 50:1213-21.

[40] Ruiz de Garibay AP, Solinis Aspiazu MA, Rodriguez Gascon A, Ganjian H, Fuchs R. Role of endocytic uptake in transfection efficiency of solid lipid nanoparticles-based nonviral vectors. J Gene Med 2013; 15:427-40.

[41] Kon E, Ad-El N, Hazan-Halevy I, Stotsky-Oterin L, Peer D. Targeting cancer with mRNA-lipid nanoparticles: key considerations and future prospects. Nat Rev Clin Oncol 2023; 20:739-54.

[42] Varkouhi AK, Scholte M, Storm G, Haisma HJ. Endosomal escape pathways for delivery of biologicals. J Controlled Release 2011; 151:220-8.

[43] Zhang M, Weng Y, Cao Z, Guo S, Hu B, Lu M, et al. ROS-activatable siRNA-engineered polyplex for NIR-triggered synergistic cancer treatment. ACS Appl Mater Interfaces 2020; 12:32289-300.

[44] Du Rietz H, Hedlund H, Wilhelmson S, Nordenfelt P, Wittrup A. Imaging small molecule-induced endosomal escape of siRNA. Nat Commun 2020; 11:1809.

[45] Li L, Zhu G, Gong C, Hu M, Tan K, Jiang L, et al. Histone demethylase KDM5D represses the proliferation, migration and invasion of hepatocellular carcinoma through the E2F1/TNNC 1 axis. Antioxid Redox Signaling 2024. doi:10.1089/ars.2023.0448.

[46] Li PP, Yan Y, Zhang HT, Cui SH, Wang CH, Wei W, et al. Biological activities of siRNA-loaded lanthanum phosphate nanoparticles on colorectal cancer. J Control Release 2020; 328:45-58.

[47] Cuesta ÁM, Palao N, Bragado P, Gutierrez-Uzquiza A, Herrera B, Sánchez A, et al. New and old key players in liver cancer. Int J Mol Sci 2023; 24:17152.

[48] He S, Cen B, Liao L, Wang Z, Qin Y, Wu Z, et al. A tumor-targeting cRGD-EGFR siRNA conjugate and its anti-tumor effect on glioblastoma in vitro and in vivo. Drug Deliv 2017; 24:471-81.

[49] Zhang X, Zhang P, An L, Sun N, Peng L, Tang W, et al. Miltirone induces cell death in hepatocellular carcinoma cell through GSDME-dependent pyroptosis. Acta Pharm Sin B 2020; 10:1397-413.

[50] Cheng YY, Qian ZY, Liu XK, Zhang YC, Chen Q, Shan TH, et al. A NIR-II photothermal nanoplatform integrating intracellular $\mathrm{Ca}^{2+}$ elevation via TRPV1 activation for enhanced antitumor therapy: a powerful combination of in situ tumor vaccination and vascular disruption. Chem Eng J 2024; 481:148274.

[51] Zhang H, Wang Y, Li M, Cao K, Qi Z, Zhu L, et al. A self-guidance biological hybrid drug delivery system driven by anaerobes to inhibit the proliferation and metastasis of colon cancer. Asian J Pharm Sci 2022; 17:892-907.

[52] Zieger-Naumann K, Kuhl F, Engele J. G protein-mediated EGFR transactivation is a common mechanism through which the CXCL12 receptors, CXCR4 and CXCR7, control human cancer cell migration. Oncol Rep 2024; 51:24.

[53] Li Z, Zhang Q, Zhang X, Jin Q, Yue Q, Li N, et al. Dihydroartemisinin inhibits melanoma migration and metastasis by affecting angiogenesis. Phytotherapy Research 2023:1-15.

[54] Xiao F, Chen Y, Qi J, Yao Q, Xie J, Jiang X. Multi-targeted peptide-modified gold nanoclusters for treating solid tumors in the liver. Adv Mater 2023; 35:e2210412.

[55] Zeng Y, Zhan YH, Liu XY, Ma JW, Liu HF, Li HR, et al. Highly efficient chemo/photothermal therapy alleviating tumor hypoxia against cancer and attenuate liver metastasis in vivo. Chem Eng J 2022; 448:137724.

[56] Patel SS, Hoogenboezem EN, Yu F, DeJulius CR, Fletcher RB, Sorets AG, et al. Core polymer optimization of ternary siRNA nanoparticles enhances in vivo safety, pharmacokinetics, and tumor gene silencing. Biomaterials 2003; 297:122098.