Cellule étiquetage et le ciblage des nanoparticules d'oxyde de fer Superparamagnetic
Summary
Targeted cell delivery is useful in a variety of biomedical applications. The goal of this protocol is to use superparamagnetic iron oxide nanoparticles (SPION) to label cells and thereby enable magnetic cell targeting approaches for a high degree of control over cell delivery and localization.
Abstract
L'administration ciblée de cellules et des agents thérapeutiques bénéficierait une large gamme d'applications biomédicales, en concentrant l'effet thérapeutique au niveau du site cible tout en minimisant les effets délétères sur les sites hors cibles. Ciblage cellulaire magnétique est une technique de livraison efficace, sûre et simple. Des nanoparticules d'oxyde de fer superparamagnétiques de (spion) sont biodégradables, biocompatibles, et peuvent être endocytose dans des cellules pour les rendre sensibles aux champs magnétiques. Le procédé de synthèse consiste à créer magnétite (Fe 3 O 4) suivie d'émulsification nanoparticules à grande vitesse pour former un poly (acide lactique-co-glycolique) (PLGA) de revêtement. Les SPIONs PLGA-magnétite sont environ 120 nm de diamètre dont le diamètre de noyau de magnétite d'environ 10 nm. Lorsqu'il est placé dans un milieu de culture, sont naturellement SPIONs endocytose par les cellules et stockées sous forme de petits agrégats à l'intérieur des endosomes cytoplasmiques. Ces particules confèrent masse magnétique suffisant pour les cellulespour permettre le ciblage à l'intérieur des champs magnétiques. Nombreux tri cellulaire et applications ciblant sont activés en rendant divers types sensibles aux champs magnétiques de cellules. SPIONs ont une variété d'autres applications biomédicales, y compris aussi bien l'utilisation comme agent de contraste pour l'imagerie médicale, l'administration de médicaments ou un gène ciblé, des analyses de diagnostic, de génération et hyperthermie locale pour le traitement des tumeurs ou la soudure de tissu.
Introduction
Targeted delivery and capture of cells to specific sites within the body is desirable for a variety of biomedical applications. Delivery of neural stem cells to the brain by MRI-guided focused ultrasound has been proposed as a possible treatment option for neurodegenerative disease, traumatic brain injury, and stroke1. Mesenchymal stem cells are being studied for their ability to deliver anti-cancer drugs to tumors due to their natural tumor-tropic properties2,3. Cardiac stem cells have been delivered to the heart as a possible treatment for myocardial infarction4,5. Vascular stents have been developed with CD34 antibodies to capture circulating progenitor cells6. While promising, these cell targeting approaches present drawbacks including lack of cell specificity, inconsistent cell retention, and off-target cell delivery.
The overall goal of the current method is to enable magnetically directed targeting of cells for a variety of cell delivery and sorting applications. Magnetic targeting allows for controlled delivery of specific cells to a specific target site with minimal off-target effects7. The magnetic fields can be generated by implanted or external devices to safely direct the movement of magnetically-labeled cells within the body8. Numerous research efforts have focused on magnetically directed targeting of stem cells to injured tissues such as the heart9-14, retina15, lung16, skin17, spinal cord18,19, bone20, liver21, and muscle22,23 in order to improve regeneration outcomes.
Magnetic targeting of cells has also been studied extensively as a means to endothelialize implantable cardiovascular devices. A uniform and complete endothelium provides a barrier between the device and circulating blood elements to mitigate thrombosis and inflammation. Endothelial cells can be delivered to the device either prior to implantation or via the vascular system following implantation. In both cases, magnetic fields are used to capture cells to the surface of the device and retain the cells when subjected to the shear stress generated by circulating blood. Magnetic vascular stents24-27 and vascular grafts28 have both been fabricated and tested for this purpose.
Magnetic cell targeting requires a strategy for labeling cells with magnetic carrier particles. These particles can be bound to the surface of cells via antibodies or ligand/receptor pairs or they can be endocytosed into the cells. Superparamagnetic iron oxide nanoparticles (SPION) are biodegradable, biocompatible, and readily endocytosed by a variety of cell types29. These particles effectively render a cell responsive to magnetic fields and are naturally degraded over time. SPIONs provide a straightforward and safe means of magnetically labeling cells in culture for a variety of magnetic targeting and sorting applications. A method for synthesizing SPIONs with a magnetite (Fe3O4) core and poly(lactic-co-glycolic acid) (PLGA) shell is provided. In addition, a method for labeling cells in culture with SPIONs is provided.
Protocol
Representative Results
Discussion
Comme pour tout protocole de synthèse de nanoparticules, la pureté des substances chimiques réactifs est critique pour la réalisation de SPIONs de haute qualité qui auront des effets cytotoxiques minimales. Il est donc important d'acheter des réactifs très purs dont l'acide oléique (≥99%), le fer (II) tétrahydrate de chlorure (≥99.99%), le fer (III) chlorure (≥99.99%), l'acétate d'éthyle (qualité HPLC, ≥99.9% ), de l'hexane (de qualité pour CLHP, ≥97.0%), de l'hydroxyde d&#…
Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors wish to acknowledge funding from the European Regional Development Fund – FNUSA-ICRC (no. CZ.1.05/ 1.1.00/ 02.0123), the American Heart Association Scientist Development Grant (AHA #06-35185N), and the National Institutes of Health (NIH #T32HL007111).
Materials
| Ammonium Hydroxide solution, 28% NH3 in H2O, ≥99.99% trace metal basis | Sigma-Aldrich | 338818-100ML | Harmful reagent – wear personal protective equipment |
| Dreschel bottle, 500 mL | Ace Glass | 5516-16 | |
| Ethyl Acetate, CHROMASOLVR Plus, for HPLC, 99.9% | Sigma-Aldrich | 650528-1L | Harmful reagent – wear personal protective equipment & work in fume hood |
| Ethyl alcohol | Sigma-Aldrich | E7023 | Harmful reagent – wear personal protective equipment |
| Filter paper, 3 cm dia, grade 1 | Fisher | 09-805P | For use with glass filter funnel |
| Glass beakers, 1 L | Fisher | FB-101-1000 | For washing SPIONs |
| Glass filter funnel, vacuum hose adapter, fits 24/40, 30 mL | Fisher | K954100-0344 | |
| Glass vial caps | Fisher | 03-391-46 | For use with glass vials |
| Glass vials, 2 mL | Fisher | 03-391-44 | For collecting magnetite gel & SPIONs |
| Hexane, CHROMASOLVR, for HPLC, ≥97.0% (GC) | Sigma-Aldrich | 34859-1L | Harmful reagent – wear personal protective equipment & work in fume hood |
| Hydrochloric acid | Sigma-Aldrich | H1758 | Harmful reagent – wear personal protective equipment & work in fume hood |
| Iron(II) chloride tetrahydrate, ≥99.99% trace metals basis | Sigma-Aldrich | 380024-5G | Harmful reagent – wear personal protective equipment |
| Iron(III) chloride anhydrous, powder, ≥99.99% trace metals basis | Sigma-Aldrich | 451649-1G | Harmful reagent – wear personal protective equipment |
| Isomantle heater, 500 mL | Voight Global | EM0500/CEX1 | |
| Laboratory mixer | Silverson | L5M-A | |
| Lyophilizer | Labconco | 7670520 | |
| Microspatulas | Fisher | 21-401-25A | For transfering magnetite gel |
| NdFeB magnet, 1 in x 1 in x 1 in | Amazing Magnets | C1000H-M | Very strong magnet, handle with care |
| Oleic acid, ≥99% (GC) | Sigma-Aldrich | O1008-5G | Store in freezer; Harmful reagent – wear personal protective equipment |
| Overhead stirrer | IKA | 2572201 | |
| Overhead stirrer clamp | IKA | 2664000 | For use with overhead stirrer |
| Overhead stirrer H-stand | IKA | 1412000 | For use with overhead stirrer |
| Phosphate buffered saline | Life Technologies | 10010-023 | |
| Plastic beakers, 250 mL | Fisher | 02-591-28 | |
| PLGA PURASORB PDLG (75/25 blend) | Purac | PDLG 7502 | PDLG 7502A may be used as well; Store in freezer |
| Pluronic F-127 powder, BioReagent, suitable for cell culture | Sigma-Aldrich | P2443-250G | |
| PTFE expandable blade paddle, 8 mm dia | SciQuip | SP4018 | |
| PTFE vessel adapter, fits 24/40, 8 mm dia paddle | Monmouth Scientific | PTFE Vessel Adaptor A480 | For use with PTFE expandable blade paddle |
| Recirculating chiller | Clarkson | 696613 | For use with rotoevaporator |
| Reflux condenser, fits 24/40, 250 mm | Ace Glass | 5997-133 | |
| Rotoevaporator | Clarkson | 216949 | |
| Round bottom flask, 50 mL, 24/40 joint | Sigma-Aldrich | Z414484 | For use with rotoevaporator |
| Rubber septa, fits 24/40 | Ace Glass | 9096-56 | |
| Separatory funnel with stopper, 250 mL | Fisher | 10-438E | |
| Sodium sulfate ACS reagent, ≥99.0%, anhydrous, granular | Sigma-Aldrich | 239313-500G | |
| Three neck round bottom flask, angled, 24/40 joints, 500 mL | Ace Glass | 6948-16 | |
| Ultrasonic cleaner perforated pan | Fisher | 15-335-20A | For use with ultrasonic cleaner |
| Ultrasonic cleaner, 2.8 L | Fisher | 15-335-20 | |
| Vacuum controller | Clarkson | 216639 | For use with rotoevaporator (optional) |
| Vacuum pump | Clarkson | 219959 | For use with rotoevaporator |
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