Streamlined miRNA loading of surface protein-specific extracellular vesicle subpopulations through electroporation
Abstract Background Extracellular vesicles (EVs) have emerged as an exciting tool for targeted delivery of therapeutics for a wide range of diseases. As nano-scale membrane-bound particles derived from living cells, EVs possess inherent capabilities as carriers of biomolecules. However, the translat...
Saved in:
| Main Authors: | , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
BMC
2024-11-01
|
| Series: | BioMedical Engineering OnLine |
| Subjects: | |
| Online Access: | https://doi.org/10.1186/s12938-024-01311-2 |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1846158317380960256 |
|---|---|
| author | Corinna Torabi Sung-Eun Choi Thomas R. Pisanic Michael Paulaitis Soojung Claire Hur |
| author_facet | Corinna Torabi Sung-Eun Choi Thomas R. Pisanic Michael Paulaitis Soojung Claire Hur |
| author_sort | Corinna Torabi |
| collection | DOAJ |
| description | Abstract Background Extracellular vesicles (EVs) have emerged as an exciting tool for targeted delivery of therapeutics for a wide range of diseases. As nano-scale membrane-bound particles derived from living cells, EVs possess inherent capabilities as carriers of biomolecules. However, the translation of EVs into viable therapeutic delivery vehicles is challenged by lengthy and inefficient processes for cargo loading and pre- and post-loading purification of EVs, resulting in limited quantity and consistency of engineered EVs. Results In this work, we develop a fast and streamlined method to load surface protein-specific subpopulations of EVs with miRNA by electroporating EVs, while they are bound to antibody-coated beads. We demonstrate the selection of CD81+ EV subpopulation using magnetic microbeads, facilitating rapid EV manipulations, loading, and subsequent purification processes. Our approach shortens the time per post-electroporation EV wash by 20-fold as compared to the gold standard EV washing method, ultracentrifugation, resulting in about 2.5-h less time required to remove unloaded miRNA. In addition, we addressed the challenge of nonspecific binding of cargo molecules due to affinity-based EV selection, lowering the purity of engineered EVs, by implementing innovative strategies, including poly A carrier RNA-mediated blocking and dissociation of residual miRNA and EV-like miRNA aggregates following electroporation. Conclusions Our streamlined method integrates magnetic bead-based selection with electroporation, enabling rapid and efficient loading of miRNA into CD81+ EVs. This approach not only achieves comparable miRNA loading efficiency to conventional bulk electroporation methods but also concentrates CD81+ EVs and allows for simple electroporation parameter adjustment, promising advancements in therapeutic RNA delivery systems with enhanced specificity and reduced toxicity. |
| format | Article |
| id | doaj-art-6c79735bf1b549dba28636b9ab6e274a |
| institution | Kabale University |
| issn | 1475-925X |
| language | English |
| publishDate | 2024-11-01 |
| publisher | BMC |
| record_format | Article |
| series | BioMedical Engineering OnLine |
| spelling | doaj-art-6c79735bf1b549dba28636b9ab6e274a2024-11-24T12:36:24ZengBMCBioMedical Engineering OnLine1475-925X2024-11-0123112310.1186/s12938-024-01311-2Streamlined miRNA loading of surface protein-specific extracellular vesicle subpopulations through electroporationCorinna Torabi0Sung-Eun Choi1Thomas R. Pisanic2Michael Paulaitis3Soojung Claire Hur4Department of Mechanical Engineering, Johns Hopkins UniversityDepartment of Mechanical Engineering, Johns Hopkins UniversityInstitute for NanoBioTechnology, Johns Hopkins UniversityCenter for Nanomedicine at Wilmer Eye Institute, Johns Hopkins University School of MedicineDepartment of Mechanical Engineering, Johns Hopkins UniversityAbstract Background Extracellular vesicles (EVs) have emerged as an exciting tool for targeted delivery of therapeutics for a wide range of diseases. As nano-scale membrane-bound particles derived from living cells, EVs possess inherent capabilities as carriers of biomolecules. However, the translation of EVs into viable therapeutic delivery vehicles is challenged by lengthy and inefficient processes for cargo loading and pre- and post-loading purification of EVs, resulting in limited quantity and consistency of engineered EVs. Results In this work, we develop a fast and streamlined method to load surface protein-specific subpopulations of EVs with miRNA by electroporating EVs, while they are bound to antibody-coated beads. We demonstrate the selection of CD81+ EV subpopulation using magnetic microbeads, facilitating rapid EV manipulations, loading, and subsequent purification processes. Our approach shortens the time per post-electroporation EV wash by 20-fold as compared to the gold standard EV washing method, ultracentrifugation, resulting in about 2.5-h less time required to remove unloaded miRNA. In addition, we addressed the challenge of nonspecific binding of cargo molecules due to affinity-based EV selection, lowering the purity of engineered EVs, by implementing innovative strategies, including poly A carrier RNA-mediated blocking and dissociation of residual miRNA and EV-like miRNA aggregates following electroporation. Conclusions Our streamlined method integrates magnetic bead-based selection with electroporation, enabling rapid and efficient loading of miRNA into CD81+ EVs. This approach not only achieves comparable miRNA loading efficiency to conventional bulk electroporation methods but also concentrates CD81+ EVs and allows for simple electroporation parameter adjustment, promising advancements in therapeutic RNA delivery systems with enhanced specificity and reduced toxicity.https://doi.org/10.1186/s12938-024-01311-2Extracellular vesiclesElectroporationEngineered extracellular vesiclesmiRNA loadingImmunopurificationTargeted gene delivery |
| spellingShingle | Corinna Torabi Sung-Eun Choi Thomas R. Pisanic Michael Paulaitis Soojung Claire Hur Streamlined miRNA loading of surface protein-specific extracellular vesicle subpopulations through electroporation BioMedical Engineering OnLine Extracellular vesicles Electroporation Engineered extracellular vesicles miRNA loading Immunopurification Targeted gene delivery |
| title | Streamlined miRNA loading of surface protein-specific extracellular vesicle subpopulations through electroporation |
| title_full | Streamlined miRNA loading of surface protein-specific extracellular vesicle subpopulations through electroporation |
| title_fullStr | Streamlined miRNA loading of surface protein-specific extracellular vesicle subpopulations through electroporation |
| title_full_unstemmed | Streamlined miRNA loading of surface protein-specific extracellular vesicle subpopulations through electroporation |
| title_short | Streamlined miRNA loading of surface protein-specific extracellular vesicle subpopulations through electroporation |
| title_sort | streamlined mirna loading of surface protein specific extracellular vesicle subpopulations through electroporation |
| topic | Extracellular vesicles Electroporation Engineered extracellular vesicles miRNA loading Immunopurification Targeted gene delivery |
| url | https://doi.org/10.1186/s12938-024-01311-2 |
| work_keys_str_mv | AT corinnatorabi streamlinedmirnaloadingofsurfaceproteinspecificextracellularvesiclesubpopulationsthroughelectroporation AT sungeunchoi streamlinedmirnaloadingofsurfaceproteinspecificextracellularvesiclesubpopulationsthroughelectroporation AT thomasrpisanic streamlinedmirnaloadingofsurfaceproteinspecificextracellularvesiclesubpopulationsthroughelectroporation AT michaelpaulaitis streamlinedmirnaloadingofsurfaceproteinspecificextracellularvesiclesubpopulationsthroughelectroporation AT soojungclairehur streamlinedmirnaloadingofsurfaceproteinspecificextracellularvesiclesubpopulationsthroughelectroporation |