Fluorescens-quenching af et liposomalt indkapslet Near-infrarøde fluorofor som et værktøj til<em> In Vivo</em> Optisk Imaging
Summary
The use of fluorophores for in vivo imaging can be greatly limited by opsonization, rapid clearance, low detection sensitivity and cytotoxic effects on the host. Encapsulation of fluorophores in liposomes by film hydration and extrusion leads to fluorescence quenching and protection which enables in vivo imaging with high detection sensitivity.
Abstract
Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous number of imaging techniques currently available and the progress in instrumentation, there is still a need for highly sensitive probes that are suitable for in vivo imaging. One typical problem of available preclinical fluorescent probes is their rapid clearance in vivo, which reduces their imaging sensitivity. To circumvent rapid clearance, increase number of dye molecules at the target site, and thereby reduce background autofluorescence, encapsulation of the near-infrared fluorescent dye, DY-676-COOH in liposomes and verification of its potential for in vivo imaging of inflammation was done. DY-676 is known for its ability to self-quench at high concentrations. We first determined the concentration suitable for self-quenching, and then encapsulated this quenching concentration into the aqueous interior of PEGylated liposomes. To substantiate the quenching and activation potential of the liposomes we use a harsh freezing method which leads to damage of liposomal membranes without affecting the encapsulated dye. The liposomes characterized by a high level of fluorescence quenching were termed Lip-Q. We show by experiments with different cell lines that uptake of Lip-Q is predominantly by phagocytosis which in turn enabled the characterization of its potential as a tool for in vivo imaging of inflammation in mice models. Furthermore, we use a zymosan-induced edema model in mice to substantiate the potential of Lip-Q in optical imaging of inflammation in vivo. Considering possible uptake due to inflammation-induced enhanced permeability and retention (EPR) effect, an always-on liposome formulation with low, non-quenched concentration of DY-676-COOH (termed Lip-dQ) and the free DY-676-COOH were compared with Lip-Q in animal trials.
Introduction
Liposomer er blevet intensivt undersøgt og tjener som en af de mest biokompatible biomedicinske medikamentleveringssystemer for kliniske anvendelser 1,2. De er hovedsageligt sammensat af phospholipider og cholesterol, som begge er biokompatible forbindelser efterligner dele af naturlige cellemembraner. Betragtninger hydrofile stoffer kan indesluttes i det vandige indre, kan lipofile midler inkorporeres i den liposomale phospholipid dobbeltlag 3. Indkapsling af stoffer i det vandige indre af liposomer giver beskyttelse mod nedbrydning in vivo og forhindrer også værtssystemet fra toksiske virkninger på cytotoksiske lægemidler, der anvendes til behandling af sygdomme, for eksempel kemoterapeutiske midler med henblik på at ødelægge tumorceller. Modifikationen af den liposomale overflade med polymerer, såsom polyethylenglycol (PEGylering) strækker sig længere den liposomale blodcirkulationen in vivo på grund af steriske 4 stabilisering. MoreovER, liposomer kan udskille høje koncentrationer af flere stoffer, såsom proteiner 5,6, hydrofile stoffer 7,8 og enzymer 9. De tjener derfor som pålidelige kliniske terapeutiske og diagnostiske værktøjer, som fortjener deres godkendelse til levering af cytotoksiske lægemidler, såsom doxorubicin til kræftbehandling 4. På grund af deres fleksibilitet, kan liposomer også fyldt med fluorochromer til diagnostiske og billed-guidede kirurgiske formål.
Fluorescensimagografi tilvejebringer en omkostningseffektiv og ikke-invasiv in vivo diagnostisk værktøj, som dog kræver nogle grundlæggende krav. Det kunne påvises, at fluorokromer, der passer bedst til in vivo imaging har karakteristisk absorption og emissionsmaksima i området, hvor lysspredning og spredning samt væv autofluorescens stammer fra vand og hæmoglobin er lav. Således sådanne prober har deres abs / em maxima mellem 650 og 900 nm 10. Udover dette, stabilitet fluorokromer både in vitro og in vivo er kritisk, da opsonisering og hurtig clearance i høj grad kan begrænse anvendelsen til in vivo imaging 11. Andre effekter såsom dårlig stabilitet og lav følsomhed eller cytotoksiske virkninger på målorganer som set med indocyaningrøn (ICG) 12-16, er uønsket og bør tages i betragtning ved udformningen af prober til in vivo billeddannelse. Disse observationer har ført til aktiv udvikling af flere prækliniske NIR fluorochromer, nanopartikler samt nye teknikker til in vivo-afbildning af inflammatoriske processer, kræft og til billedbehandling-guidet kirurgi 17-20. Trods stabilitet mest præklinisk NIRF (nær-infrarød fluorescens) farvestoffer in vitro, deres hurtige perfusion og clearance gennem leveren og nyren hindre deres anvendelse i in vivo optisk billeddannelse af sygdomme og inflammatoriske processer.
ntent "> Vi har derfor præsentere en protokol til indkapsling af fluorokromer såsom godt karakteriseret nærinfrarøde fluorescerende farvestof DY-676-COOH, kendt for sin tendens til selv-dæmpningen ved relativt høje koncentrationer 21 i liposomer. Ved høje koncentrationer H- dimerdannelse og / eller PI-stabling interaktioner mellem fluoroforen molekyler beliggende inden hinandens Förster radius resultat i Forster resonansenergioverførsel (FRET) mellem de fluorochrom molekyler. ved lav koncentration i rummet mellem fluoroforen molekyler stiger, og derved forhindre pi-stabling interaktion og H-dimerdannelse og resulterer i høj fluorescensemission. Kontakten mellem høj og lav koncentration og den ledsagende fluorescensundertrykkelse og aktivering er en lovende strategi, der kan udnyttes til optisk billeddannelse 22. I denne henseende, indkapsling af høje koncentrationer af NIRF farvestof DY-676-COOH i det vandige indre af liposomer er flere favorable til in vivo billeddannelse end det frie farvestof. Udfordringen af metoden ligger først og fremmest i den korrekte indkapsling og for det andet, i valideringen af de fordele, der følger af at indkapsle høje koncentrationer af farvestoffet. Sammenligning af billeddannende egenskaber bratkølede liposomer med den frie farvestof og også med en liposomformulering ikke-standset med lave koncentrationer af farvestoffet er uundværlig. Vi viser ved en simpel, men meget effektiv film hydrering og ekstrudering protokol kombineret med alternative fryse og tø-cykler, som indkapsling af quenching koncentrationer af DY-676-COOH i liposomer er muligt. Andre fremgangsmåder anvendes til at fremstille liposomer, såsom omvendt fase inddampning metode 23 samt ethanol injektion metode 24 aktivere liposompræparat med høje indkapslingseffektiviteter for mange hydrofile stoffer. Imidlertid er arten af det stof, der skal indkapsles, kan påvirke indkapslingseffektiviteten. I realitetenfilmen hydrering og ekstrudering protokol præsenteres her viste den højeste effektivitet til indkapsling af DY-676-COOH. For at illustrere fordelene ved liposomal indkapsling af DY-676-COOH, en zymosan-induceret ødem model, der tillader undersøgelse af inflammatoriske processer inden for et par timer blev anvendt. Her er det vist, at liposomer med høje koncentrationer af det indkapslede DY-676-COOH er mere egnede til hele kroppen in vivo optisk billeddannelse af inflammatoriske processer end det frie farvestof eller den liposomale formulering ikke-quenchet med lave farvestofkoncentrationer. Således den underliggende protokol giver en enkel og hurtig metode til at producere standset fluorescerende liposomer og validering af deres aktivering og billedbehandling potentiale både in vitro og in vivo.Protocol
Representative Results
Discussion
Da liposomer også kan tjene som fremføringssystemer til fluorescerende farvestoffer, de gør det muligt billeddannelse af target sygdomme. Indkapslingen af høje koncentrationer af fluorescerende farvestoffer, såsom NIRF farvestof DY676-COOH anvendes her, fører til et højt niveau af fluorescens quenching af den indesluttede farvestof. Fluorescens quenching, et fænomen ses med mange fluoroforer i høj koncentration kan udnyttes i flere in vivo imaging applikationer, hvor en høj følsomhed og pålide…
Declarações
The authors have nothing to disclose.
Acknowledgements
Dette arbejde blev støttet af Deutsche Forschungsgemeinschaft tilskud HI-698 / 10-1 og RU-1652 / 1-1. Vi takker Doreen maj for fremragende teknisk bistand og selskabet DYOMICS GmbH, Jena for deres venlige støtte.
Materials
| Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
| Materials and equipments for preparation of liposomes | |||
| egg phospahtidylcholine | Avanti Polar Lipids | 840051P | Dissolve in Chloroform and store in glass vials (214 mg/ml) |
| cholesterol | Sigma | C8667 | Dissolve in Chloroform and store in glass vials (134 mg/ml) |
| 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt) | Avanti Polar Lipids | 880120P | Dissolve in Chloroform and store in glass vials (122 mg/ml) |
| 1,2-dioleoyl-snglycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (ammonium salt) | Avanti Polar Lipids | 810145P | Dissolve in Chloroform and store in glass vials (2mg/ml) |
| Sartorius MC1 (d = 0.01 mg) | Sartorius AG | Research RC 210 P | used for weighing the phospholipids |
| Rotavapor | Büchi Labortechnik AG | R-114 | used for hydration of phospholipid film |
| Waterbath | Büchi Labortechnik AG | R-481 | used for hydration of phospholipid film |
| Vacuum Controller | Büchi Labortechnik AG | B-720 | used for hydration of phospholipid film |
| Vacobox | Büchi Labortechnik AG | B-177 | used for hydration of phospholipid film |
| Circulation Chiller | LAUDA DR. R. WOBSER GMBH & CO. KG |
WKL 230 | used for hydration of phospholipid film |
| DY-676-COOH | Dyomics GmbH | 676-00 | Dissolve in 10 mM Tris and store stock at -20°C |
| Tris-(Hydroxymethyl)-aminomethan | Applichem | A1086 | buffer 10 mM, pH 7.4 |
| Trichlormethan | Carl Roth GmbH + Co. KG | Y015.2 | used for liposome preparation |
| Sonicator | Merck Eurolab GmbH | USR 170 H | used for liposome preparation |
| Vortex Genie 2 (Pop-off Cup, No. 146-3011-00) | Scientific Industries Inc. | SI-0256 | used for liposome preparation |
| Sephadex G25 medium | GE Healthcare Europe GmbH | 17-0033-01 | used for liposome purification |
| Triton X100 | Ferak Berlin GmbH | 505002 | used to destruct liposomes for dye quantification |
| LiposoFast-Basic | Avestin Inc. | used for the extrusion of liposomes | |
| Polycarbonate filter membrane, 100 nm (Whatman Nucleopore Trans Etch Membrane, NUCLEPR PC 19 MM, 0.1 U) | VWR | used for the extrusion of liposomes via LiposoFast-Basic | |
| Fluostar Optima | BMG Labtech | used for dye quantification | |
| Zetasizer Nano ZS | Malvern | used for the determination of liposome size and zetapotential | |
| Ultracentrifuge | Beckmann Coulter GmbH | XL 80 | used for concentration of the samples |
| Rotor | Beckmann Coulter GmbH | SW 55 TI | used for concentration of the samples |
| Materials and equipments for the evaluation of liposome and optical imaging | |||
| Zymosan-A from Saccharomyces cereviciae | Sigma | Z4250-250MG | used for induction of inflammation |
| Isotonic Saline (0.9) | Fresenius GmbH | PZN-2159621 | used for the dilution of Zymosan-A |
| Isoflurane vaporizer | Ohmeda Isotec 4 | used for anesthesizing animals | |
| Isoflurane | Actavis GmbH | PZN-7253744 | anesthesia |
| Thermo Mat Pro 20 W | Lucky Reptile | 61202-HTP-20 | used to keep animals warm during anesthesia |
| Omnican-F (1 ml injection) | Braun | PZN-3115465 | used for subcutaneous and intravenous application of probes |
| Panthenol eye cream | Jenapharm | PZN-3524531 | used to prevent dryness of the eyes of animals during anesthesia |
| Hanks buffered saline solution | PAA Laboratories /Biochrom AG | L2045 | w/o Mg2+, Ca2+ and phenol red. For dilution of probes and for washing of cells |
| 8-Well chamber slides | BD Biosciences | 354108 | used for cell culture followed by microscopy |
| Cell culture flasks | Greiner BioOne | ||
| Cell culture media | Gibco (life technologies GmbH) | ||
| Fetal calf serum | Invitrogen | ||
| Poly-L-Lysine solution (0,01% – 50 ml) | Sigma | P4832 | used to coat cell culture chamber slides |
| Mountant Permafluor | ThermoScientific | S21022-3 | Mounting solution for microscopy |
| Hoechst-33258 | AppliChem | DNA stain for microscopy | |
| Hera-Safe | Heraeus Instruments | sterile work bench used for cell culture | |
| HERA cell | Heraeus Instruments | Incubator used for cell culture | |
| LSM510-Meta | Zeiss | used for confocal microscopy | |
| Maestro-TM in vivo fluorescence imaging system | CRi, Woburn | used for whole body fluorescence imaging of small animals | |
| Spectrophotometer (Ultrospec 4300 pro UV) | GE Healthcare | used for measurement of absorption | |
| Spectrofluorometer (Jasco FP-6200) | Jasco | used for measurement of fluorescence emission | |
| Animals | |||
| NMRI mice (8-12 weeks old, male) | Elevage Janvier, France | used for inflammation trials | |
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