An erythrocyte membrane-fused plant-derived nanoparticles as a gene therapy vehicle for the treatment of CI/R injury

Abstract

Ischemic stroke is currently the second leading cause of death worldwide, and insufficient endogenous neurogenesis is the greatest cause of post-stroke disability. MicroRNAs have been proven to hold therapeutic potential, unfortunately, they have a low stability that hinders their clinical usage. Our earlier work revealed that Panax notoginseng derived exosome like nanoparticles, namely PDNs have potential to bypass BBB and reduce the cerebral ischemia/reperfusion (CI/R) damage. In this study, we employed microRNA-124 as a model therapeutic gene, utilizing its engineered variant Agomir-124 (Ago124) to optimize loading efficiency. The therapeutic effects of Ago124@R-PDN were further assessed in several sets of experiments. Pharmacokinetic study showed that erythrocyte membrane extended the half-life of PDNs from 7 min to 11.3 h, and the loading efficiency of Ago124 reached 40%. In an in vitro oxygen-glucose deprivation/reperfusion (OGD/R) model, Ago124@R-PDN enhanced IL-10 production in microglia by 67% (vs 11.7% with free Ago124), and promoted Tuj1⁺ neuronal differentiation by 2.23-fold compared with vehicle. Also, Ago124@R-PDN brought gene cargo into the brain, alleviated infarct volume, and improved functional behaviors in model mice. At last, we demonstrated that surface glycosyl of PDN facilitated its brain-entering ability by being recognized by sodium-glucose linked transporter-1 protein. In conclusion, our erythrocyte fused PDNs offer a promising strategy for delivering biomacromolecule to treat brain diseases.

Keywords

Microrna-124 / Ischemic stroke / Plant-derived exosome like nanoparticles / Gene therapy / Erythrocyte membrane

Conflicts of interest

The authors have no conflicts of interest to declare. All co-authors have seen and agree with the contents of the manuscript and there is no financial interest to report. We certify that the submission is original work and is not under review at any other publication.

Acknowledgment

This work was supported by National Natural Science Foundation of China (82374296, 82271965, 62331021), Development Project of Shanghai Peak Disciplines-Integrated Medicine (201801), ShanghaiMunicipalScienceand Technology Major Project (2018SHZDZX01) and Shanghai Municipal Science and Technology Explorer Project (23TS1400500).

Supplementary materials

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.ajps.2025.101089. The figures and tables with "S" before the serial number are included in the Supplementary material.

References

[1] Feigin VL, Brainin M, Norrving B, Martins SO, Pandian J, Lindsay P, et al. World stroke organization: global stroke fact sheet 2025. Int J Stroke 2025; 20(2):132-44.

[2] Rupaimoole R, Han HD, Lopez-Berestein G, Sood AK. MicroRNA therapeutics: principles, expectations, and challenges. Chin J Cancer 2011; 30(6):368-70.

[3] Lichun C, Pastrana E, Tavazoie M, Doetsch F. miR-124 regulates adult neurogenesis in the subventricular zone stem cell niche. Nat. Neurosci. 2009; 12(4):399-408.

[4] Doeppner TR, Doehring M, Bretschneider E, Zechariah A, Kaltwasser B, Müller B. MicroRNA-124 protects against focal cerebral ischemia via mechanisms involving Usp14-dependent rest degradation. Acta Neuropathol. 2013; 126(2):251-65.

[5] Ponomarev ED, Veremeyko T, Barteneva N, Krichevsky AM, Weiner HL. MicroRNA-124 promotes microglia quiescence and suppresses eae by deactivating macrophages via the C/EBP- a-PU. 1 pathway. Nat Med 2011; 17(1):64-70.

[6] Dasgupta I, Chatterjee A. Recent advances in miRNA delivery systems. Methods Protoc 2021; 4(1):10.

[7] ELA S, Mäger I, Breakefield XO, Wood MJ. Extracellular vesicles: biology and emerging therapeutic opportunities. Nat Rev Drug Discov 2013; 12(5):347-57.

[8] Wang C, Xu M, Fan Q, Li C, Zhou X. Therapeutic potential of exosome-based personalized delivery platform in chronic inflammatory diseases. Asian J Pharm Sci 2023; 18(1):100772.

[9] Yuzhou Z, Tangrong W, Jianxin Z, Sen W, Zhijing W, Jiali L, et al. Plant-derived nanovesicles: a promising frontier in tissue repair and antiaging. J. Agric. Food Chem. 2025; 73(22):13159-77.

[10] Sarwareddy KK, Singh AD, Patnam S, Sesuraj BA, Ponamgi S, Thakur BK, et al. Harnessing tomato-derived small extracellular vesicles as drug delivery system for cancer therapy. Future Sci OA 2025; 11(1):2461956.

[11] Shiyi L, Ru Z, Anni W, Yang L, Miaomiao Z, Kim J. Panax notoginseng: derived exosome-like nanoparticles attenuate ischemia reperfusion injury via altering microglia polarization. J Nanobiotechnol 2023; 21(1): 416.

[12] Matsumoto A, Takahashi Y, Hsinyi C, Yiwen W, Yamamoto A, Ishihama Y, et al. Blood concentrations of small extracellular vesicles are determined by a balance between abundant secretion and rapid clearance. J Extracell Vesicles 2020; 9(1):1696517.

[13] Dehaini D, Wei X, Fang RH, Masson S, Angsantikul P, Luk BT, et al. Erythrocyte-platelet hybrid membrane coating for enhanced nanoparticle functionalization. Adv Mater 2017; 29(16):1606209.

[14] Qin J, Zimiao L, Yongzhi M, Peng Y, Haibao P, Ranran G, et al. Red blood cell membrane-camouflaged melanin nanoparticles for enhanced photothermal therapy. Biomaterials 2017; 143:29-45.

[15] Cheng L, Xiaoquan Y, Jie A, Kai C, Xiaolin H, xiaoshuai Z, et al. Red blood cell membrane-enveloped O₂ self-supplementing biomimetic nanoparticles for tumor imaging-guided enhanced sonodynamic therapy. Theranostics 2020; 10(2):867-79.

[16] Kimiz-Gebologlu I, Oncel SS. Exosomes: large-scale production, isolation, drug loading efficiency, and biodistribution and uptake. J Control Release 2022; 347:533-43.

[17] Balaraman AK, Babu MA, Moglad E, Mandaliya V, Rekha MM, Gupta S, et al. Exosome-mediated delivery of crispr-cas9: a revolutionary approach to cancer gene editing. Pathol Res Pract 2025; 266:155785.

[18] Greene MA, Worley GA, Udoka ANS, Powell RR, Bruce T, Klotz JL, et al. Use of agomir and antagomir technologies to alter satellite cell proliferation in vitro, miRNA expression, and muscle fiber hypertrophy in intrauterine growth-restricted lambs. Front Mol Biosci 2023; 10:1286890.

[19] Kim M, Lee Y, Lee M. Hypoxia-specific anti-rage exosomes for nose-to-brain delivery of anti-mir-181a oligonucleotide in an ischemic stroke model. Nanoscale 2021; 13(33):14166-78.

[20] Yuwei H, Ruixiang L, Haichun L, Shuya Z, Wentao D, Qian W, et al. Erythroliposomes: integrated hybrid nanovesicles composed of erythrocyte membranes and artificial lipid membranes for pore-forming toxin clearance. ACS Nano 2019; 13(4):4148-59.

[21] Siling D, Shanshan X, Xiangjuan D, Tifei Y, Bo P, Yanxia R. Primary microglia isolation from postnatal mouse brains. J Vis Exp 2021; 168:e62237.

[22] Turner RC, DiPasquale K, Logsdon AF, Tan Z, Naser ZJ, Huber JD, et al. The role for infarct volume as a surrogate measure of functional outcome following ischemic stroke. J Syst Integr Neurosci 2016; 2(4). doi:10.15761/JSIN.1000136.

[23] Bermpohl D, You Z, Lo EH, Kim HH, Whalen MJ. TNF alpha and fas mediate tissue damage and functional outcome after traumatic brain injury in mice. J Cereb Blood Flow Metab 2007; 27(11):1806-18.

[24] Yuan W, Rehana KL, Guodong C. Microglia-mediated neuroinflammation and neuroplasticity after stroke. Front Cell Neurosci 2022; 16:980722.

[25] Weizhuo L, Jiyue W. Crosstalk among glial cells in the blood-brain barrier injury after ischemic stroke. Mol Neurobiol 2024; 61(9):6161-74.

[26] Po H, Hui L, Wenshuo S, Hongzheng W, Xiaoxuan Y, Yingjie Q, et al. Cholesterol-associated lysosomal disorder triggers cell death of hematological malignancy: dynamic analysis on cytotoxic effects of LW-218. Acta Pharm Sin B 2021; 11(10):3178-92.

[27] Nishida-Aoki N, Tominaga N, Kosaka N, Ochiya T. Altered biodistribution of deglycosylated extracellular vesicles through enhanced cellular uptake. J Extracell Vesicles 2020; 9(1):1713527.

[28] Patching SG. Glucose transporters at the blood-brain barrier: function, regulation and gateways for drug delivery. Mol Neurobiol 2017; 54(2):1046-77.

[29] Elfeber K, Köhler A, Lutzenburg M, Osswald C, Galla HJ, Witte OW, et al. Localization of the Na⁺-d-glucose cotransporter SGLT 1 in the blood-brain barrier. Histochem Cell Biol 2004; 121(3):201-7.

[30] Khare P, Edgecomb SX, Hamadani CM, Tanner EEL, S Manickam D. Lipid nanoparticle-mediated drug delivery to the brain. Adv. Drug Deliv. Rev. 2023; 197:114861.

[31] Johnsen KB, Burkhart A, Melander F, Kempen PJ, Vejlebo JB, Siupka P, et al. Targeting transferrin receptors at the blood-brain barrier improves the uptake of immunoliposomes and subsequent cargo transport into the brain parenchyma. Sci Rep 2017; 7(1):10396.

[32] Kumari S, Ahsan SM, Kumar JM, Kondapi AK, Rao NM. Overcoming blood brain barrier with a dual purpose temozolomide loaded lactoferrin nanoparticles for combating glioma (SERP-17-12433). Sci Rep 2017; 7(1):6602.

[33] Xiong RR, Ling GX, Zhang YQ, Guan JB, Zhang P. Nucleic acid delivery by ionizable nanocarriers for brain disease treatment. Brain-X 2023; 1:e7.

[34] Bischof D, Cornetta K. Flexibility in cell targeting by pseudotyping lentiviral vectors. Methods Mol Biol 2010; 614:53-68.

[35] Burger C, Gorbatyuk OS, Velardo MJ, Peden CS, Williams P, Zolotukhin S, et al. Recombinant aav viral vectors pseudotyped with viral capsids from serotypes 1, 2, and 5 display differential efficiency and cell tropism after delivery to different regions of the central nervous system. Mol Ther 2004; 10(2):302-17.