Methods, Mechanisms, and Application Prospects for Enhancing Extracellular Vesicle Uptake

Abstract

Extracellular vesicles (EVs) are considered to be a new generation of bioinspired nanoscale drug delivery systems due to their low immunogenicity, natural functionality, and excellent biocompatibility. However, limitations such as low uptake efficiency, insufficient production, and inhomogeneous performance undermine their potential. To address these issues, numerous researchers have put forward various methods and applications for enhancing EV uptake in recent decades. In this review, we introduce various methods for the cellular uptake of EVs and summarize recent advances on the methods and mechanisms for enhancing EV uptake. In addition, we provide further understanding regarding enhancing EV uptake and put forward prospects and challenges for the development of EV-based therapy in the future.

Keywords

extracellular vesicles / exosomes / extracellular vesicle uptake / extracellular vesicle-based therapy

References

[1] PegtelDM, GouldSJ. Exosomes. Annu Rev Biochem, 2019, 88(1): 487-514

[2] El AndaloussiS, MägerI, BreakefieldXO, et al.. Extracellular vesicles: biology and emerging therapeutic opportunities. Nat Rev Drug Discov, 2013, 12(5): 347-357

[3] EttelaieC, CollierMEW, MaraveyasA, et al.. Characterization of physical properties of tissue factor-containing microvesicles and a comparison of ultracentrifuge-based recovery procedures. J Extracell Vesicles, 2014, 3(1): 1

[4] BattistelliM, FalcieriE. Apoptotic Bodies: Particular Extracellular Vesicles Involved in Intercellular Communication. Biology, 2020, 9(1): 21

[5] ElmoreS. Apoptosis: A Review of Programmed Cell Death. Toxicol Pathol, 2007, 35(4): 495-516

[6] MathieuM, Martin-JaularL, LavieuG, et al.. Specificities of secretion and uptake of exosomes and other extracellular vesicles for cell-to-cell communication. Nat Cell Biol, 2019, 21(1): 9-17

[7] ColomboM, RaposoG, TheryC. Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles. Annu Rev Cell Dev Biol, 2014, 30: 255-289

[8] MaasSLN, BreakefieldXO, WeaverAM. Extracellular Vesicles: Unique Intercellular Delivery Vehicles. Trends Cell Biol, 2017, 27(3): 172-188

[9] AbelsER, BreakefieldXO. Introduction to Extracellular Vesicles: Biogenesis, RNA Cargo Selection, Content, Release, and Uptake. Cell Mol Neurobiol, 2016, 36(3): 301-312

[10] Nolte-’T HoenENM, BuschowSI, AndertonSM, et al.. Activated T cells recruit exosomes secreted by dendritic cells via LFA-1. Blood, 2009, 113(9): 1977-1981

[11] ZhangB, WangM, GongA, et al.. HucMSC-Exosome Mediated-Wnt4 Signaling Is Required for Cutaneous Wound Healing. Stem Cells, 2015, 33(7): 2158-2168

[12] CuiX, HeZ, LiangZ, et al.. Exosomes From Adipose-derived Mesenchymal Stem Cells Protect the Myocardium Against Ischemia/Reperfusion Injury Through Wnt/β-Catenin Signaling Pathway. J Cardiovasc Pharmacol, 2017, 70(4): 225-231

[13] RatajczakJ, MiekusK, KuciaM, et al.. Embryonic stem cell-derived microvesicles reprogram hematopoietic progenitors: evidence for horizontal transfer of mRNA and protein delivery. Leukemia, 2006, 20(5): 847-856

[14] MenY, YelickJ, JinS, et al.. Exosome reporter mice reveal the involvement of exosomes in mediating neuron to astroglia communication in the CNS. Nat Commun, 2019, 10(1): 4136

[15] BangC, BatkaiS, DangwalS, et al.. Cardiac fibroblast-derived microRNA passenger strand-enriched exosomes mediate cardiomyocyte hypertrophy. J Clin Invest, 2014, 124(5): 2136-2146

[16] ZamaniP, FereydouniN, ButlerAE, et al.. The therapeutic and diagnostic role of exosomes in cardiovascular diseases. Trends Cardiovasc Med, 2019, 29(6): 313-323

[17] HowittJ, HillAF. Exosomes in the Pathology of Neurodegenerative Diseases. J Biol Chem, 2016, 291(52): 26589-26597

[18] ChenN, SunXY, DingZC, et al.. Small Extracellular Vesicles Secreted by Peri-urethral Tissues Regulate Fibroblast Function and Contribute to the Pathogenesis of Female Stress Urinary Incontinence. Curr Med Sci, 2023, 43(4): 803-810

[19] DuanCY, FanWL, ChenF. Roles of Optineurin and Extracellular Vesicles in Glaucomatous Retinal Cell Loss. Curr Med Sci, 2023, 43(2): 367-375

[20] OsakiM, OkadaF. Exosomes and Their Role in Cancer Progression. Yonago Acta Med, 2019, 62(2): 182-190

[21] DengZ, LiuY, LiuC, et al.. Immature myeloid cells induced by a high-fat diet contribute to liver inflammation. Hepatology, 2009, 50(5): 1412-1420

[22] NakaseI, KobayashiNB, Takatani-NakaseT, et al.. Active macropinocytosis induction by stimulation of epidermal growth factor receptor and oncogenic Ras expression potentiates cellular uptake efficacy of exosomes. Sci Rep, 2015, 5(1): 10300

[23] NakaseI, FutakiS. Combined treatment with a pH-sensitive fusogenic peptide and cationic lipids achieves enhanced cytosolic delivery of exosomes. Sci Rep, 2015, 5(1): 10112

[24] XuH, LiaoC, LiangS, et al.. A Novel Peptide-Equipped Exosomes Platform for Delivery of Antisense Oligonucleotides. ACS Appl Mater Interfaces, 2021, 13(9): 10760-10767

[25] Martínez-SantillánA, González-ValdezJ. Novel Technologies for Exosome and Exosome-like Nanovesicle Procurement and Enhancement. Biomedicines, 2023, 11(5): 1487

[26] TranNH, NguyenDD, NguyenNM, et al.. Dualtargeting exosomes for improved drug delivery in breast cancer. Nanomedrcme (Lond), 2023, 18(7): 599-611

[27] LuanX, SansanaphongprichaK, MyersI, et al.. Engineering exosomes as refined biological nanoplatforms for drug delivery. Acta Pharmacol Sin, 2017, 38(6): 754-763

[28] TianT, WangY, WangH, et al.. Visualizing of the cellular uptake and intracellular trafficking of exosomes by live-cell microscopy. J Cell Biochem, 2010, 111(2): 488-496

[29] TianT, ZhuYL, HuFH, et al.. Dynamics of exosome internalization and trafficking. J Cell Physiol, 2013, 228(7): 1487-1495

[30] SokolovaV, LudwigA, HornungS, et al.. Characterisation of exosomes derived from human cells by nanoparticle tracking analysis and scanning electron microscopy. Colloids and Surfaces B: Biointerfaces, 2011, 87(1): 146-150

[31] TakahashiY, NishikawaM, ShinotsukaH, et al.. Visualization and in vivo tracking of the exosomes of murine melanoma B16-BL6 cells in mice after intravenous injection. J Biotechnol, 2013, 165(2): 77-84

[32] van NielG, D’AngeloG, RaposoG. Shedding light on the cell biology of extracellular vesicles. Nat Rev Mol Cell Biol, 2018, 19(4): 213-228

[33] GyorgyB, HungME, BreakefieldXO, et al.. Therapeutic applications of extracellular vesicles: clinical promise and open questions. Annu Rev Pharmacol Toxicol, 2015, 55: 439-464

[34] GurungS, PerocheauD, TouramanidouL, et al.. The exosome journey: from biogenesis to uptake and intracellular signalling. Cell Commun Signal, 2021, 19(1): 47

[35] ParoliniI, FedericiC, RaggiC, et al.. Microenvironmental pH Is a Key Factor for Exosome Traffic in Tumor Cells. J Biol Chem, 2009, 284(49): 34211-34222

[36] MontecalvoA, LarreginaAT, ShufeskyWJ, et al.. Mechanism of transfer of functional microRNAs between mouse dendritic cells via exosomes. Blood, 2012, 119(3): 756-766

[37] MorelliAE, LarreginaAT, ShufeskyWJ, et al.. Endocytosis, intracellular sorting, and processing of exosomes by dendritic cells. Blood, 2004, 104(10): 3257-3266

[38] EscreventeC, KellerS, AltevogtP, et al.. Interaction and uptake of exosomes by ovarian cancer cells. BMC Cancer, 2011, 11(1): 108

[39] AraldiRP, DelvalleDA, Da CostaVR, et al.. Exosomes as a Nano-Carrier for Chemotherapeutics: A New Era of Oncology. Cells, 2023, 12(17): 2144

[40] GondaA, KabagwiraJ, SenthilGN, et al.. Internalization of Exosomes through Receptor-Mediated Endocytosis. Mol Cancer Res, 2019, 17(2): 337-347

[41] MckelveyKJ, PowellKL, AshtonAW, et al.. Exosomes: Mechanisms of Uptake. J Circ Biomark, 2015, 4: 7

[42] MettlenM, ChenP, SrinivasanS, et al.. Regulation of Clathrin-Mediated Endocytosis. Annu Rev Biochem, 2018, 87(1): 871-896

[43] KissAL, BotosE. Endocytosis via caveolae: alternative pathway with distinct cellular compartments to avoid lysosomal degradation?. J Cell Mol Med, 2009, 13(7): 1228-1237

[44] BarresC, BlancL, Bette-BobilloP, et al.. Galectin-5 is bound onto the surface of rat reticulocyte exosomes and modulates vesicle uptake by macrophages. Blood, 2010, 115(3): 696-705

[45] NanboA, KawanishiE, YoshidaR, et al.. Exosomes derived from Epstein-Barr virus-infected cells are internalized via caveola-dependent endocytosis and promote phenotypic modulation in target cells. J Virol, 2013, 87(18): 10334-10347

[46] MenckK, KlemmF, GrossJC, et al.. Induction and transport of Wnt 5a during macrophage-induced malignant invasion is mediated by two types of extracellular vesicles. Oncotarget, 2013, 4(11): 2057-2066

[47] Izquierdo-UserosN, Naranjo-GómezM, ArcherJ, et al.. Capture and transfer of HIV-1 particles by mature dendritic cells converges with the exosome-dissemination pathway. Blood, 2009, 113(12): 2732-2741

[48] NabiIR, LePU. Caveolae/raft-dependent endocytosis. The Journal of Cell Biology, 2003, 161(4): 673-677

[49] DohertyGJ, McmahonHT. Mechanisms of endocytosis. Annu Rev Biochem, 2009, 78: 857-902

[50] FengD, ZhaoW, YeY, et al.. Cellular Internalization of Exosomes Occurs Through Phagocytosis. Traffic, 2010, 11(5): 675-687

[51] GordonS. Phagocytosis: An Immunobiologic Process. Immunity, 2016, 44(3): 463-475

[52] LimJP, GleesonPA. Macropinocytosis: an endocytic pathway for internalising large gulps. Immunol Cell Biol, 2011, 89(8): 836-843

[53] HeusermannW, HeanJ, TrojerD, et al.. Exosomes surf on filopodia to enter cells at endocytic hot spots, traffic within endosomes, and are targeted to the ER. J Cell Biol, 2016, 213(2): 173-184

[54] MattilaPK, LappalainenP. Filopodia: molecular architecture and cellular functions. Nat Rev Mol Cell Biol, 2008, 9(6): 446-454

[55] LehmannMJ, ShererNM, MarksCB, et al.. Actin- and myosin-driven movement of viruses along filopodia precedes their entry into cells. J Cell Biol, 2005, 170(2): 317-325

[56] BarrèsC, BlancL, Bette-BobilloP, et al.. Galectin-5 is bound onto the surface of rat reticulocyte exosomes and modulates vesicle uptake by macrophages. Blood, 2010, 115(3): 696-705

[57] FitznerD, SchnaarsM, van RossumD, et al.. Selective transfer of exosomes from oligodendrocytes to microglia by macropinocytosis. J Cell Sci, 2011, 124(3): 447-458

[58] FrühbeisC, FröhlichD, KuoWP, et al.. Neurotransmitter-Triggered Transfer of Exosomes Mediates Oligod-endrocyte-Neuron Communication. PLoS Biol, 2013, 11(7): e1001604

[59] NanboA, KawanishiE, YoshidaR, et al.. Exosomes Derived from Epstein-Barr Virus-Infected Cells Are Internalized via Caveola-Dependent Endocytosis and Promote Phenotypic Modulation in Target Cells. J Virol, 2013, 87(18): 10334-10347

[60] SvenssonKJ, ChristiansonHC, WittrupA, et al.. Exosome Uptake Depends on ERK1/2-Heat Shock Protein 27 Signaling and Lipid Raft-mediated Endocytosis Negatively Regulated by Caveolin-1. J Biol Chem, 2013, 288(24): 17713-17724

[61] RejmanJ, OberleV, ZuhornIS, et al.. Size-dependent internalization of particles via the pathways of clathrin- and caveolae-mediated endocytosis. Biochem J, 2004, 377: 159-169 Pt 1)

[62] McmahonHT, BoucrotE. Molecular mechanism and physiological functions of clathrin-mediated endocytosis. Nat Rev Mol Cell Biol, 2011, 12(8): 517-533

[63] WangZ, TiruppathiC, ChoJ, et al.. Delivery of nanoparticle: complexed drugs across the vascular endothelial barrier via caveolae. IUBMB Life, 2011, 63(8): 659-667

[64] AkincA, BattagliaG. Exploiting endocytosis for nanomedicines. Cold Spring Harb Perspect Biol, 2013, 5(11): a16980

[65] Costa VerderaH, Gitz-FrancoisJJ, SchiffelersRM, et al.. Cellular uptake of extracellular vesicles is mediated by clathrin-independent endocytosis and macropinocytosis. J Control Release, 2017, 266: 100-108

[66] YeH, WangF, XuG, et al.. Advancements in engineered exosomes for wound repair: current research and future perspectives. Front Bioeng Biotechnol, 2023, 11: 1301362

[67] NakagawaY, ArafilesJVV, KawaguchiY, et al.. Stearylated Macropinocytosis-Inducing Peptides Facilitating the Cellular Uptake of Small Extracellular Vesicles. Bioconjug Chem, 2022, 33(5): 869-880

[68] NakaseI, NoguchiK, FujiiI, et al.. Vectorization of biomacromolecules into cells using extracellular vesicles with enhanced internalization induced by macropinocytosis. Sci Rep, 2016, 6(1): 34937

[69] HiraseS, AokiA, HattoriY, et al.. Dodecaborate-Encapsulated Extracellular Vesicles with Modification of Cell-Penetrating Peptides for Enhancing Macropinocytotic Cellular Uptake and Biological Activity in Boron Neutron Capture Therapy. Mol Pharm, 2022, 19(4): 1135-1145

[70] NoguchiK, ObukiM, SumiH, et al.. Macropinocytosis-Inducible Extracellular Vesicles Modified with Antimicrobial Protein CAP18-Derived Cell-Penetrating Peptides for Efficient Intracellular Delivery. Mol Pharm, 2021, 18(9): 3290-3301

[71] ShimodaA, MiuraR, TatenoH, et al.. Assessment of Surface Glycan Diversity on Extracellular Vesicles by Lectin Microarray and Glycoengineering Strategies for Drug Delivery Applications. Small Methods, 2022, 6(2): e2100785

[72] Nishida-AokiN, TominagaN, KosakaN, et al.. Altered biodistribution of deglycosylated extracellular vesicles through enhanced cellular uptake. J Extracell Vesicles, 2020, 9(1): 1713527

[73] ZhanQ, YiK, LiX, et al.. Phosphatidylcholine-Engineered Exosomes for Enhanced Tumor Cell Uptake and Intracellular Antitumor Drug Delivery. Macromol Biosci, 2021, 21(8): e2100042

[74] ZouJ, ShiM, LiuX, et al.. Aptamer-Functionalized Exosomes: Elucidating the Cellular Uptake Mechanism and the Potential for Cancer-Targeted Chemotherapy. Anal Chem, 2019, 91(3): 2425-2430

[75] HadeMD, SuireCN, SuoZ. An Effective Peptide-Based Platform for Efficient Exosomal Loading and Cellular Delivery of a microRNA. ACS Appl Mater Interfaces, 2023, 15(3): 3851-3866

[76] LiaoZ, LiuH, MaL, et al.. Engineering Extracellular Vesicles Restore the Impaired Cellular Uptake and Attenuate Intervertebral Disc Degeneration. ACS Nano, 2021, 15(9): 14709-14724

[77] MeyerC, LosaccoJ, StickneyZ, et al.. Pseudotyping exosomes for enhanced protein delivery in mammalian cells. Int J Nanomedicine, 2017, 12: 3153-3170

[78] TemchuraVV, TenbuschM, NchindaG, et al.. Enhancement of immunostimulatory properties of exosomal vaccines by incorporation of fusion-competent G protein of vesicular stomatitis virus. Vaccine, 2008, 26(29): 3662-3672

[79] HungME, LeonardJN. Stabilization of Exosometargeting Peptides via Engineered Glycosylation. J Biol Chem, 2015, 290(13): 8166-8172

[80] ZhangJ, SongH, DongY, et al.. Surface Engineering of HEK293 Cell-Derived Extracellular Vesicles for Improved Pharmacokinetic Profile and Targeted Delivery of IL-12 for the Treatment of Hepatocellular Carcinoma. Int J Nanomedicine, 2023, 18: 209-223

[81] KelemenA, CarmiI, OszvaldÁ, et al.. IFITM1 expression determines extracellular vesicle uptake in colorectal cancer. Cell Mol Life Sci, 2021, 78(21–22): 7009-7024

[82] ZhangS, GuoM, GuoT, et al.. DAL-1/4.1B promotes the uptake of exosomes in lung cancer cells via Heparan Sulfate Proteoglycan 2 (HSPG2). Mol Cell Biochem, 2022, 477(1): 241-254

[83] HazawaM, TomiyamaK, Saotome-NakamuraA, et al.. Radiation increases the cellular uptake of exosomes through CD29/CD81 complex formation. Biochem Biophys Res Commun, 2014, 446(4): 1165-1171

[84] MizutaR, SasakiY, KawasakiR, et al.. Magnetically Navigated Intracellular Delivery of Extracellular Vesicles Using Amphiphilic Nanogels. Bioconjug Chem, 2019, 30(8): 2150-2155

[85] GongC, ZhangX, ShiM, et al.. Tumor Exosomes Reprogrammed by Low pH Are Efficient Targeting Vehicles for Smart Drug Delivery and Personalized Therapy against their Homologous Tumor. Adv Sci (Weinh), 2021, 8(10): 2002787

[86] KimH, KangJ, MunD, et al.. Calcium chloride enhances the delivery of exosomes. PLoS One, 2019, 14(7): e220036

[87] Ferrero-AndrésA, ClosaD, Roselló-CatafauJ, et al.. Polyethylene Glycol 35 (PEG35) Modulates Exosomal Uptake and Function. Polymers (Basel), 2020, 12(12): 3044

[88] MaS, SongL, BaiY, et al.. Improved intracellular delivery of exosomes by surface modification with fluorinated peptide dendrimers for promoting angiogenesis and migration of HUVECs. RSC Adv, 2023, 13(17): 11269-11277

[89] MatsukiY, YanagawaT, SumiyoshiH, et al.. Modification of exosomes with carbonate apatite and a glycan polymer improves transduction efficiency and target cell selectivity. Biochem Biophys Res Commun, 2021, 583: 93-99

[90] TakenakaT, NakaiS, KatayamaM, et al.. Effects of gefitinib treatment on cellular uptake of extracellular vesicles in EGFR-mutant non-small cell lung cancer cells. Int J Pharm, 2019, 572: 118762

[91] ZhaoZ, McgillJ, Gamero-KubotaP, et al.. Microfluidic on-demand engineering of exosomes towards cancer immunotherapy. Lab Chip, 2019, 19(10): 1877-1886

[92] BuiS, DancourtJ, LavieuG. Virus-Free Method to Control and Enhance Extracellular Vesicle Cargo Loading and Delivery. ACS Appl Bio Mater, 2023, 6(3): 1081-1091

[93] WuP, TangY, JinC, et al.. Neutrophil membrane engineered HucMSC sEVs alleviate cisplatin-induced AKI by enhancing cellular uptake and targeting. J Nanobiotechnology, 2022, 20(1): 353

[94] HuS, WangX, LiZ, et al.. Platelet membrane and stem cell exosome hybrids enhance cellular uptake and targeting to heart injury. Nano Today, 2021, 39: 101210

[95] ShaoJ, ZaroJ, ShenY. Advances in Exosome-Based Drug Delivery and Tumor Targeting: From Tissue Distribution to Intracellular Fate. Int J Nanomedicine, 2020, 15: 9355-9371

[96] GondaA, KabagwiraJ, SenthilGN, et al.. Exosomal survivin facilitates vesicle internalization. Oncotarget, 2018, 9(79): 34919-34934

[97] TkachM, KowalJ, ZucchettiAE, et al.. Qualitative differences in T-cell activation by dendritic cell-derived extracellular vesicle subtypes. The EMBO Journal, 2017, 36(20): 3012-3028

[98] GuanS, LiQ, LiuP, et al.. Experimental immunology Umbilical cord blood-derived dendritic cells loaded with BGC823 tumor antigens and DC-derived exosomes stimulate efficient cytotoxic T-lymphocyte responses and antitumor immunity in vitro and in vivo. Cent Eur J Immunol, 2014, 2(2): 142-151

[99] Sobo-VujanovicA, MunichS, VujanovicNL. Dendritic-cell exosomes cross-present Toll-like receptor-ligands and activate bystander dendritic cells. Cell Immunol, 2014, 289(1–2): 119-127

[100] MunichS, Sobo-VujanovicA, BuchserWJ, et al.. Dendritic cell exosomes directly kill tumor cells and activate natural killer cells via TNF superfamily ligands. Oncoimmunology, 2014, 1(7): 1074-1083

[101] ZhengY, TuC, ZhangJ, et al.. Inhibition of multiple myeloma-derived exosomes uptake suppresses the functional response in bone marrow stromal cell. Int J Oncol, 2019, 54(3): 1061-1070

[102] BuschowSI, NolteT, HoenENM, Van NielG, et al.. MHC II in Dendritic Cells is Targeted to Lysosomes or T Cell-Induced Exosomes Via Distinct Multivesicular Body Pathways. Traffic, 2009, 10(10): 1528-1542

[103] VerweijFJ, BebelmanMP, JimenezCR, et al.. Quantifying exosome secretion from single cells reveals a modulatory role for GPCR signaling. J Cell Biol, 2018, 217(3): 1129-1142

[104] HoribeS, TanahashiT, KawauchiS, et al.. Mechanism of recipient cell-dependent differences in exosome uptake. BMC Cancer, 2018, 18(1): 47

[105] EguchiS, TakefujiM, SakaguchiT, et al.. Cardiomyocytes capture stem cell-derived, anti-apoptotic microRNA-214 via clathrin-mediated endocytosis in acute myocardial infarction. J Biol Chem, 2019, 294(31): 11665-11674

[106] BenmerahA, BayrouM, Cerf-BensussanN, et al.. Inhibition of clathrin-coated pit assembly by an Eps15 mutant. J Cell Sci, 1999, 112(9): 1303-1311

[107] YoonJH, AshktorabH, SmootDT, et al.. Uptake and tumor-suppressive pathways of exosome-associated GKN1 protein in gastric epithelial cells. Gastric Cancer, 2020, 23(5): 848-862

[108] KoumangoyeRB, SakweAM, GoodwinJS, et al.. Detachment of Breast Tumor Cells Induces Rapid Secretion of Exosomes Which Subsequently Mediate Cellular Adhesion and Spreading. PLoS One, 2011, 6(9): e24234

[109] SkotlandT, HessvikNP, SandvigK, et al.. Exosomal lipid composition and the role of ether lipids and phosphoinositides in exosome biology. J Lipid Res, 2019, 60(1): 9-18