通过注射药物加载的脂体将药物定位到拉瓦尔斑马鱼巨噬细胞
Summary
在这里，我们描述了药物加载脂体及其显微注射到幼虫斑马鱼的合成，目的是将药物输送到巨噬细胞。
Abstract
斑马鱼 （Danio rerio） 幼虫已经发展成为一个流行的模型，研究宿主-病原体相互作用和先天免疫细胞对炎症性疾病的贡献，由于其功能保守的先天免疫系统.它们还广泛用于研究先天免疫细胞如何帮助指导发育过程。通过利用幼虫斑马鱼的光学透明度和遗传可采性，这些研究通常侧重于活成像方法，以功能特征表荧光标记巨噬细胞和中性粒细胞在完整的动物。由于其在疾病发病机制中各异质性和不断扩大的作用，巨噬细胞受到广泛关注。除了基因操作，化学干预现在经常被用来操纵和检查幼虫斑马鱼的巨噬细胞行为。这些药物的输送通常仅限于通过直接浸泡或显微注射被动地瞄准免费药物。这些方法依赖于以下假设：对巨噬细胞行为的任何变化都是药物对巨噬细胞本身的直接影响的结果，而不是对另一种细胞类型的直接影响的下游后果。在这里，我们提出我们的方案，通过显微注射药物加载荧脂体，专门针对幼斑马鱼巨噬细胞的药物。我们揭示，波罗夏默188改性药物加载蓝色荧光脂质体很容易被巨噬细胞，而不是中性粒细胞。我们还提供了证据，证明以这种方式交付的药物可以按照药物作用机制的方式影响巨噬菌体活动。这项技术对于研究人员来说，如果能够确保药物针对巨噬细胞，当药物毒性太大，无法通过浸泡等传统方法输送时，这项技术将有价值的。
Introduction
单核噬菌体系统为抵御入侵病原体提供了第一道防线。该系统由单核细胞、单核衍生树突细胞和巨噬细胞组成，它们对外来病原体进行积极的噬菌体，从而限制病原体的传播。除了这些噬菌体和微生物效应器功能外，树突状细胞和巨噬细胞还能够产生细胞因子和抗原，以激活适应性免疫系统1。在这些细胞中，巨噬细胞由于功能异质性不同，并参与多种炎症性疾病而受到特别关注，从自身免疫和传染病到癌症2、3、4、5、6、7。巨噬细胞的可塑性及其在功能上适应组织环境需求的能力，需要采用实验方法直接观察和询问体内的这些细胞。
幼虫斑马鱼是研究体内巨噬细胞功能和可塑性的理想模型生物。幼虫斑马鱼的光学透明度为直接观察巨噬细胞的行为提供了一个窗口，特别是当与巨噬细胞标记转基因报告线结合时。利用幼虫斑马鱼的活成像潜力和实验可采性，对与人类疾病9、10、11、12、13、14、15直接相关的巨噬菌体功能提出了许多重要的见解。其中许多研究还利用斑马鱼药物活性的高保护（一个属性，支撑了它们作为整个动物药物发现平台16，17，18），利用化学干预药理作用操纵巨噬细胞功能。迄今为止，这些药理治疗主要通过浸泡（要求药物是水溶性的）或直接微注射免费药物（图1A）来提供。这些被动分娩策略的局限性包括脱靶效应和一般毒性，可能妨碍评估对巨噬菌体功能的任何影响。此外，在调查药物对巨噬细胞的影响时，不知道药物是作用于巨噬细胞本身或通过更间接的机制。在进行类似的化学干预研究以研究巨噬细胞功能时，我们认识到，开发一种廉价而直接的传递方法，专门针对巨噬细胞的药物，是未满足的。
脂质体是微观的，生物相容性，脂质双层囊泡，可以封装蛋白质，核苷酸和药物货物19。脂质体的单层或多层脂质双层结构形成水性内流明，其中水溶性药物可以结合，而疏水性药物可以集成到脂质膜中。此外，脂质体的物理化学性质，包括大小，电荷和表面修改可以操纵，以定制其靶向到特定的细胞20，21。脂体的这些特点，使他们成为一个有吸引力的工具，提供药物，并提高目前的治疗方案20的精度。由于脂质体是由巨噬细胞自然噬菌体（一种利用其常规使用，专门用于向巨噬细胞进行消融实验的结节功能的一种特征22），它们作为巨噬细胞特异性药物输送的一个有吸引力的选择（图1B）。
该协议描述了药物配方成蓝色荧光脂质体涂覆与亲水性聚合物波罗沙默188，在脂质体表面形成保护层，并已证明，以提高药物保留，并具有优越的生物相容性23。Poloxamer被选择用于脂质体的表面涂层，因为我们先前的研究表明，与聚乙烯乙二醇改性脂质体相比，Poloxamer修饰的脂质体在静脉注射大鼠爪和类似的药代动力学后，在兔子的皮下注射后表现出更好的生物相容性23。还介绍了其向幼虫斑马鱼和活成像进行显微注射的规程，以评估其巨噬细胞靶向能力和细胞内腔内的定位，这些细胞是脂质降解和细胞质药物输送所必需的。作为概念验证，我们以前曾使用这种技术来瞄准两种药物，以巨噬细胞来抑制其激活在幼虫斑马鱼模型急性痛风炎症24。这种药物输送技术扩展了斑马鱼研究人员的化学”工具包”，这些研究人员希望确保其感兴趣的药物的巨噬菌体靶向。
Protocol
1. 制备药物装的玛丽娜蓝标记脂体 注：携带蓝色荧光染料、Marina Blue 和药物的脂质体使用薄膜水化方法制备，后插入了 poloxamer 188。除非另有规定，否则所有程序均在室温下进行。控制脂体只携带滨海蓝色和PBS。此处的示例描述了使用线粒体靶向抗氧化药物25加载脂体，该药物在代表性结果中用作概念验证。 为了制备脂质体，溶解16.4毫克（22.2μmol）?…
Representative Results
本文描述的用于制备荧光脂质体封闭药物的薄膜水化方法是一种简单而经济高效的方法。根据本研究所使用的方案，脂体有望成为单层23、24。表1总结了所生产的脂质体的大小、齐塔潜力、药物载荷和诱捕效率。脂体粒体（药物加载前后）的粒径相似（表1）。与对照脂质体相比，药物载脂质体的表面电荷（Zeta电位）略为中性，…
Discussion
在这里，我们提供了一个详细的协议，以制定药物加载的脂体，专门针对幼虫斑马鱼的巨噬细胞。该方法可用于剖析巨噬细胞在某些疾病模型中的作用，确保直接有针对性地向巨噬细胞提供药物。此外，当药物的一般毒性限制其使用时，如浸泡等更常规的路线，可以使用。此处描述的协议提供了用于靶向幼虫斑马鱼中先天免疫细胞的其他纳米颗粒系统的替代方案。这些措施包括将抗疟药物利法平?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了向C.J.H.（新西兰卫生研究理事会和马斯登基金、新西兰皇家学会）和Z.W.（奥克兰大学学院研究发展基金）提供的赠款的支持。作者感谢阿尔哈德·马哈贡卡尔对斑马鱼设施、生物医学成像研究室、奥克兰大学医学院的辅助成像和格雷厄姆·利施克（Graham Lieschke）赠送Tg（mpeg1：EGFP）记者专线的专家管理。
Materials
| 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) | Avanti Polar Lipids, Inc. | 850355P | |
| 1,2-diseteroyl-sn-glycero-3-phosphocholine (DSPE) | Avanti Polar Lipids, Inc. | 850367P | |
| 1.0 µm Whatman Nuclepore Track-Etched polycarbonate membranes | GE Healthcare Life Sciences | 110610 | |
| 25 mL round-bottom flask | Sigma-Aldrich | Z278262 | |
| 35 mm culture dish | Thermo Scientific | 150460 | |
| Acetonitrile | Sigma-Aldrich | 34998 | |
| Agilent 1260 Infinity Diode Array Detector | Agilent Technologies | G4212B | |
| Agilent 1260 Infinity Quaternary Pump | Agilent Technologies | G1311B | |
| Agilent 1290 Infinity Series Thermostat | Agilent Technologies | G1330B | |
| Avanti mini-extruder Avanti Polar Lipids Inc. | Avanti Polar Lipids Inc. | ||
| borosilicate microinjection needles | Warner Instruments | 203-776-0664 | |
| CaCl2 | Sigma-Aldrich | C4901-100G | |
| cholesterol | Sigma-Aldrich | C8667 | |
| Dumont No.5 fine tip forceps | Fine Science Tools | 11251-10 | |
| Eppendorf Microloader pipette tip | Eppendorf | 5242956003 | |
| Eppendorf SmartBlock 1.5 mL, thermoblock for 24 reaction vessels | Eppendorf | 4053-6038 | |
| eyelash manipulator | Ted Pella Inc. | 113 | |
| hemocytometer | Hawksley | BS.748 | |
| HEPES | BDH Chemicals | 441474J | |
| HPLC system | Agilent Technologies | 1260 series HPLC system | |
| KCl | Sigma-Aldrich | P9541-1KG | |
| low melting point agarose | Invitrogen | 16520-100 | |
| LysoTracker Deep Red | Invitrogen | L12492 | 1 mM stock solution in DMSO, keep at -20 °C and protect from light. |
| LysoTracker Deep Red | Thermo Scientific | L12492 | |
| magnetic stand | Narishige | GJ-1 | |
| Marina Blue 1,2-dihexadecanoyl-sn-glycero-phosphoethanolamine (Marina Blue DHPE) | Invitrogen | M12652 | Keep at -20 °C and protect from light. |
| Methanol | Sigma-Aldrich | 34860 | |
| methyl cellulose | Sigma-Aldrich | M0387-500G | |
| methylene blue | Alfa Aesar | 42771 | |
| MgSO4 | Sigma-Aldrich | 230391-500G | |
| micromanipulator | Narishige | M-152 | |
| mineral oil | Sigma-Aldrich | M-3516 | |
| Mitochondria-targeting antioxidant MitoTEMPO | Sigma-Aldrich | SML0737 | |
| MitoSOX Red Mitochondrial Superoxide Indicator | Thermo Scientific | M36008 | |
| MitoTEMPO | Sigma-Aldrich | SML0737 | Keep at -20 °C and protect from light. |
| N-Phenylthiourea (PTU) | Sigma-Aldrich | P7629-10G | Take care when handling, toxic. |
| NaCl | BDH Chemicals | 27810.295 | |
| PBS (pH 7.4) | Gibco | 10010-023 | |
| Petri dish (100 mm x 20 mm) | Corning Inc. | 430167 | |
| Phenomenex C18 Gemini-NZ 3 mm 250 mm x 4.6 mm column | Phenomenex | 00G-4439-E0 | |
| pHrodo Red Escherichia coli BioParticles Conjugate | Thermo Scientific | P35361 | |
| pHrodo Red Escherichia coli BioParticles Conjugate | Invitrogen | P35361 | Keep at -20 °C and protect from light. Make 1 mg/mL stock solution by dissolving 2 mg lyophilized product in 2 mL of PBS supplemented with 20 mM HEPES, pH 7.4. |
| plastic transfer pipette | Medi'Ray | RL200C | |
| poloxamer 188 | BASF Corporation | ||
| pressure injector | Applied Scientific Instruments | MPPI-2 | |
| rotary evaporator | Büchi, Flawil, Switzerland | Büchi R-215 Rotavapor | |
| Scanning confocal microscope | Olympus | Olympus FV1000 FluoView | |
| Sorvall WX+ Ultracentrifuge | Thermo Scientific | 75000090 | |
| stereomicroscope | Leica | MZ12 | |
| Tricaine | Sigma-Aldrich | A5040-25G | Make 4 mg/mL stock solution (in deionzed H2O) and keep at -20 °C. |
| triton-X100 | Sigma-Aldrich | X100-100ML | |
| Ultrasonic bath | Thermo Scientific | FB-11205 | |
| Volocity Image Analysis Software | PerkinElmer | version 6.3 | |
| water bath | |||
| Zetasizer Nano | Malvern Instruments Ltd | Zetasizer Nano ZS ZEN 3600 |
References
- Chow, A., Brown, B. D., Merad, M. Studying the mononuclear phagocyte system in the molecular age. Nature Reviews Immunology. 11 (11), 788-798 (2011).
- Li, Q., Barres, B. A. Microglia and macrophages in brain homeostasis and disease. Nature Reviews Immunology. 18 (4), 225-242 (2018).
- Krenkel, O., Tacke, F. Liver macrophages in tissue homeostasis and disease. Nature Reviews Immunology. 17 (5), 306-321 (2017).
- Alderton, G. K. Tumour immunology: turning macrophages on, off and on again. Nature Reviews Immunology. 14 (3), 136-137 (2014).
- Moore, K. J., Sheedy, F. J., Fisher, E. A. Macrophages in atherosclerosis: a dynamic balance. Nature Reviews Immunology. 13 (10), 709-721 (2013).
- Lawrence, T., Natoli, G. Transcriptional regulation of macrophage polarization: enabling diversity with identity. Nature Reviews Immunology. 11 (11), 750-761 (2011).
- Chawla, A., Nguyen, K. D., Goh, Y. P. Macrophage-mediated inflammation in metabolic disease. Nature Reviews Immunology. 11 (11), 738-749 (2011).
- Renshaw, S. A., Trede, N. S. A model 450 million years in the making: zebrafish and vertebrate immunity. Disease models and mechanisms. 5 (1), 38-47 (2012).
- Hall, C. J., et al. Immunoresponsive gene 1 augments bactericidal activity of macrophage-lineage cells by regulating beta-oxidation-dependent mitochondrial ROS production. Cell Metabolism. 18 (2), 265-278 (2013).
- Hall, C. J., et al. Blocking fatty acid-fueled mROS production within macrophages alleviates acute gouty inflammation. Journal of Clinical Investigation. 125 (5), 1752-1771 (2018).
- Cambier, C. J., et al. Mycobacteria manipulate macrophage recruitment through coordinated use of membrane lipids. Nature. 505 (7482), 218-222 (2014).
- Davis, J. M., et al. Real-time visualization of mycobacterium-macrophage interactions leading to initiation of granuloma formation in zebrafish embryos. Immunity. 17 (6), 693-702 (2002).
- Madigan, C. A., et al. A Macrophage Response to Mycobacterium leprae Phenolic Glycolipid Initiates Nerve Damage in Leprosy. Cell. 170 (5), 973-985 (2017).
- Tobin, D. M., et al. The lta4h locus modulates susceptibility to mycobacterial infection in zebrafish and humans. Cell. 140 (5), 717-730 (2010).
- Volkman, H. E., et al. Tuberculous granuloma induction via interaction of a bacterial secreted protein with host epithelium. Science. 327 (5964), 466-469 (2010).
- Bowman, T. V., Zon, L. I. Swimming into the future of drug discovery: in vivo chemical screens in zebrafish. ACS Chemical Biology. 5 (2), 159-161 (2010).
- Kaufman, C. K., White, R. M., Zon, L. Chemical genetic screening in the zebrafish embryo. Nature Protocols. 4 (10), 1422-1432 (2009).
- Zon, L. I., Peterson, R. T. In vivo drug discovery in the zebrafish. Nature Reviews Drug Discovery. 4 (1), 35-44 (2005).
- Malam, Y., Loizidou, M., Seifalian, A. M. Liposomes and nanoparticles: nanosized vehicles for drug delivery in cancer. Trends in Pharmacological Sciences. 30 (11), 592-599 (2009).
- Torchilin, V. P. Recent advances with liposomes as pharmaceutical carriers. Nature Reviews Drug Discovery. 4 (2), 145-160 (2005).
- Immordino, M. L., Dosio, F., Cattel, L. Stealth liposomes: review of the basic science, rationale, and clinical applications, existing and potential. International Journal of Nanomedicine. 1 (3), 297-315 (2006).
- Astin, J. W., et al. Innate immune cells and bacterial infection in zebrafish. Methods in Cell Biology. 138, 31-60 (2017).
- Zhang, W., et al. Post-insertion of poloxamer 188 strengthened liposomal membrane and reduced drug irritancy and in vivo precipitation, superior to PEGylation. Journal of Controlled Release. 203, 161-169 (2015).
- Wu, Z., et al. Liposome-Mediated Drug Delivery in Larval Zebrafish to Manipulate Macrophage Function. Zebrafish. 16 (2), 171-181 (2019).
- Cader, M. Z., et al. C13orf31 (FAMIN) is a central regulator of immunometabolic function. Nature Immunology. 17 (9), 1046-1056 (2016).
- Chono, S., Tanino, T., Seki, T., Morimoto, K. Influence of particle size on drug delivery to rat alveolar macrophages following pulmonary administration of ciprofloxacin incorporated into liposomes. Journal of Drug Targeting. 14 (8), 557-566 (2006).
- Chono, S., Tanino, T., Seki, T., Morimoto, K. Uptake characteristics of liposomes by rat alveolar macrophages: influence of particle size and surface mannose modification. Journal of Pharmact and Pharmacology. 59 (1), 75-80 (2007).
- Chono, S., Tauchi, Y., Morimoto, K. Influence of particle size on the distributions of liposomes to atherosclerotic lesions in mice. Drug Development and Industrial Pharmacy. 32 (1), 125-135 (2006).
- Rosen, J. N., Sweeney, M. F., Mably, J. D. Microinjection of zebrafish embryos to analyze gene function. Journal of Visualized Experiments. (25), (2009).
- Hall, C., Flores, M. V., Crosier, K., Crosier, P. Live cell imaging of zebrafish leukocytes. Methods in Molecular Biology. 546, 255-271 (2009).
- Kapellos, T. S., et al. A novel real time imaging platform to quantify macrophage phagocytosis. Biochemical Pharmacology. 116, 107-119 (2016).
- Shen, K., Sidik, H., Talbot, W. S. The Rag-Ragulator Complex Regulates Lysosome Function and Phagocytic Flux in Microglia. Cell Reports. 14 (3), 547-559 (2016).
- Ellett, F., Pase, L., Hayman, J. W., Andrianopoulos, A., Lieschke, G. J. mpeg1 promoter transgenes direct macrophage-lineage expression in zebrafish. Blood. 117 (4), 49-56 (2011).
- Hall, C., Flores, M. V., Storm, T., Crosier, K., Crosier, P. The zebrafish lysozyme C promoter drives myeloid-specific expression in transgenic fish. BMC Developmental Biology. 7, 42 (2007).
- Ahsan, F., Rivas, I. P., Khan, M. A., Torres Suarez, A. I. Targeting to macrophages: role of physicochemical properties of particulate carriers–liposomes and microspheres–on the phagocytosis by macrophages. Journal of Controlled Release. 79 (1-3), 29-40 (2002).
- Martin, W. J., Walton, M., Harper, J. Resident macrophages initiating and driving inflammation in a monosodium urate monohydrate crystal-induced murine peritoneal model of acute gout. Arthritis and Rheumatology. 60 (1), 281-289 (2009).
- Faires, J. S., McCarty, D. J. Acute arthritis in man and dog after intrasynovial injection of sodium urate crystals. Lancet. 280, 682-685 (1962).
- Martin, W. J., Harper, J. L. Innate inflammation and resolution in acute gout. Immunology and Cell Biology. 88 (1), 15-19 (2010).
- Fenaroli, F., et al. Nanoparticles as drug delivery system against tuberculosis in zebrafish embryos: direct visualization and treatment. ACS Nano. 8 (7), 7014-7026 (2014).
- Robertson, J. D., Ward, J. R., Avila-Olias, M., Battaglia, G., Renshaw, S. A. Targeting Neutrophilic Inflammation Using Polymersome-Mediated Cellular Delivery. Journal of Immunology. 198 (9), 3596-3604 (2017).
- Le Guellec, D., Morvan-Dubois, G., Sire, J. Y. Skin development in bony fish with particular emphasis on collagen deposition in the dermis of the zebrafish (Danio rerio). International Journal of Developmental Biology. 48 (2-3), 217-231 (2004).
- Kelly, C., Jefferies, C., Cryan, S. A. Targeted liposomal drug delivery to monocytes and macrophages. Journal of Drug Delivery. 2011, 727241 (2011).
- Fidler, I. J., et al. Design of liposomes to improve delivery of macrophage-augmenting agents to alveolar macrophages. 암 연구학. 40 (12), 4460-4466 (1980).
- Ng, A. N., et al. Formation of the digestive system in zebrafish: III. Intestinal epithelium morphogenesis. Developmenal Biology. 286 (1), 114-135 (2005).
Cite This Article
Linnerz, T., Kanamala, M., Astin, J. W., Dalbeth, N., Wu, Z., Hall, C. J. Targeting Drugs to Larval Zebrafish Macrophages by Injecting Drug-Loaded Liposomes. J. Vis. Exp. (156), e60198, doi:10.3791/60198 (2020).