荧光淬一个脂质体包封的近红外荧光团的作为一个工具<em>在体内</em>光学成像
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
脂质体已被广泛研究，并作为最生物相容性生物医学的药物递送系统的临床应用1,2之一。它们主要由磷脂和胆固醇，这两者都是生物相容的化合物模仿天然细胞膜份。而亲水性物质可在含水内部被截留，亲脂性试剂可以脂质体磷脂双层3内被引入。脂质体的含水内部中的封装的物质给予保护，防止降解在体内 ，也防止了主机系统从用于疾病的治疗的细胞毒性药物，例如化学治疗旨在破坏肿瘤细胞的毒性作用。脂质体表面与像聚乙二醇聚合物的修饰（PEG化）进一步延伸体内的脂质体的血液循环时间，由于空间位稳定4。 Moreov呃脂质体可隔离的高浓度的几种物质，如蛋白质5,6，亲水性物质7,8和酶9。因此，它们作为可靠的临床治疗和诊断工具，值得他们的批准用于递送细胞毒性药物，如阿霉素治疗癌症4。由于其灵活性，脂质体还可以装载有荧光染料用于诊断和图像引导手术目的。
荧光成像提供了一个高性价比的和非侵入体内诊断工具，但需要一些基本要求。它可以证明，这适合最适合于体内成像的荧光染料具有特征吸收和发射最大值在光分散和散射，以及组织自发荧光从水始发和血红蛋白是低的范围内。因此，这样的探针具有其650和900纳米之间10 ABS /青霉最大值。除此之外，无论是在体外和体内的荧光染料的稳定性是至关重要的，如调理作用和快速清除可大大限制了它们用于体内成像应用11。其他效果，例如差的稳定性和低灵敏度或观察与吲哚花青绿（ICG）12-16对靶器官的细胞毒作用，是不希望的，并用于体内成像设计探针时，必须考虑到。这些观察导致了若干临床前的近红外荧光染料，纳米颗粒以及用于体内成像的炎性过程，癌症和用于图像引导手术17-20新技术的积极发展。尽管大多数的临床前NIRF（近红外荧光）的稳定性的染料在体外 ，其通过肝脏和肾脏快速灌注和清除阻碍它们在疾病和炎症过程的体内光学成像的使用。
ntent“>因此，我们提出了一个协议对荧光染料的封装，如良好表征的近红外荧光染料DY-676-COOH，在脂质体以其倾向自淬火，在相对 高浓度的21，在高浓度的H-二聚体形成和/或PI-层叠荧光团分子的增加之间位于彼此的福斯特半径结果在荧光分子之间荧光共振能量转移（FRET）内的荧光团的分子之间的相互作用，在低浓度的空间中，从而防止PI-堆积作用和H-二聚体形成并导致高的荧光发射。高，低浓度和伴随荧光猝灭和激活之间的开关是一种很有前途的策略，可被利用为光学成像22上 。在这方面，封装的高浓度的NIRF染料的DY-676-COOH在脂质体的含水内部更发vorable用于体内成像比游离染料。该方法的挑战在于在正确的封装首先和其次，在从包封高浓度的染料产生的利益的验证。与游离染料与非淬火脂质体制剂与低浓度的染料的比较猝灭脂质体的成像特性，也是必不可少的。我们显示通过简单的，但高度有效膜水化和挤压协议结合交替冻结和解冻循环是DY-676-COOH的淬火浓度的脂质体中的封装是可行的。用于制备脂质体的其它方法，如逆相蒸发法23以及乙醇注入法24使脂质体制剂具有高包封率的许多亲水性的物质。然而，要被封装的物质的性质可以影响所述包封效率。实际上，这里介绍的薄膜水化和挤压协议揭示了DY-676-COOH的封装效率最高。为了说明DY-676-COOH，一个酵母多糖诱发的水肿模型，它允许在几个小时内的炎症过程的研究的脂质体包封的好处，被使用。这里，它被证明脂质体与高浓度的包封的DY-676-COOH是在炎症过程中比游离染料或未淬火脂质体制剂具有低染料浓度的体内光学成像更适合于全身。因而底层协议提供了一种简单而快速的方法，以产生荧光猝灭脂质体和在体外和体内的活化和成像潜在的有效性。Protocol
注：所有的程序都是由区域动物委员会，并按照有关伦理使用动物的国际准则认可。 1.准备材料和仪器自发形成囊泡分散液的制备（SFV） 溶解和制备原液以下磷脂：214毫克/毫升的蛋磷脂酰胆碱（EPC）134毫克/毫升胆固醇，122毫克/毫升1,2- distearoyl- SN -glycero -3- phosphoethanolamine- -N – [甲氧基（聚乙二醇）-2000]（铵盐）（MPEG 2000 -DSPE）和2毫克/毫升…
Representative Results
高浓度的荧光染料，如这里所使用的脂质体的含水内部的NIRF染料DY676-COOH的封装导致荧光猝灭的一个高的水平。荧光猝灭，这种现象可见有许多荧光团以高浓度，可以利用几种体内成像应用，其中一个高灵敏度的目标区域的可靠的检测要求较高。使用脂质体还提供了保护，这是不可缺少的用于体内应用的染料。脂质体的彻底的表征是必要的，包括几个因素，如染料包封，稳定性和脂?…
Discussion
因为脂质体还可以作为递送系统用于荧光染料，它们使目标疾病的成像。高浓度的荧光染料如NIRF染料，这里使用DY676-COOH的封装导致截留染料的荧光淬灭的高水平。荧光猝灭，这种现象以高浓度看到的许多荧光团可被利用在几种体内成像应用，其中，具有高灵敏度的目标区域的可靠的检测被征求。使用脂质体还提供了保护，这是不可缺少的用于体内应用的染料。
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Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作是由德意志研究联合会授予 HI-698 / 10-1和RU-1652 / 1-1的支持。我们感谢琳五月优秀的技术援助和公司DYOMICS有限公司，耶拿的盛情支持。
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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Citar este artigo
Tansi, F. L., Rüger, R., Rabenhold, M., Steiniger, F., Fahr, A., Hilger, I. Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging. J. Vis. Exp. (95), e52136, doi:10.3791/52136 (2015).