Name: targeting drugs to larval zebrafish macrophages by injecting drug-loaded liposomes
Uploaded: 2020-02-18T13:00:00.000+00:00
Duration: 11 min 31 s
Description: Watch this Scientific Journal Video about Targeting Drugs to Larval Zebrafish Macrophages by Injecting Drug-Loaded Liposomes at JoVE.com

This work was supported by grants awarded to C.J.H. (Health research Council of New Zealand and Marsden Fund, Royal Society of New Zealand) and Z.W. (Faculty Research Development Fund from the University of Auckland). The authors thank Alhad Mahagaonkar for expert management of the zebrafish facility, the Biomedical Imaging Research Unit, School of Medical Sciences, University of Auckland for assistance with confocal imaging and Graham Lieschke for gifting the Tg(mpeg1:EGFP) reporter line.

1. Preparation of Drug-loaded Marina Blue-labeled Liposomes
NOTE: Liposomes carrying the blue fluorescent dye, Marina Blue and drug are prepared using a thin film hydration method with post insertion of poloxamer 188. All procedures are performed at room temperature unless otherwise specified. Control liposomes only carry Marina blue and PBS. The example here describes loading liposomes with a mitochondria-targeting antioxidant drug25 that is used in the representative results as a proof-of-concept.
<ol>
	<li>To prepare liposomes, dissolve 16.4 mg (22.2 &#181;mol) of the phospholipids 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (see Table of Materials), 4.3 mg (11.1 &#181;mol) cholesterol (see Table of Materials) and 2.8 mg (3.7 &#181;mol) 1,2-diseteroyl-sn-glycero-3-phosphocholine (see Table of Materials, in 3 mL of chloroform:methanol (3:1 v/v) in a 25 mL round-bottom flask.</li>
	<li>To the above mixture, add 31 nmol of Marina Blue 1,2-dihexadecanoyl-sn-glycero-phosphoethanolamine (see Table of Materials) and 300 nmol of a mROS inhibiting drug (see Table of Materials) and sonicate briefly to fully dissolve. For control liposomes, only add Marina Blue 1,2-dihexadecanoyl-sn-glycero-phosphoethanolamine. 
	NOTE: To avoid photobleaching of the light-sensitive Marina Blue fluorescent dye, protect all procedures from light, where possible. This is also important if the drug to be loaded is light-sensitive.</li>
	<li>Remove the solvent slowly on a rotary evaporator (see Table of Materials) over a temperature controlled water bath set to 45 &#176;C by rotating at 75 rpm under vacuum pressure (200 mbar for 15 min then 0 mbar for 20 min). This forms a dry thin lipid film within the glass walls of the flask that is further dried for 45 min under N2 to facilitate complete removal of the organic solvent.</li>
	<li>Following formation of the thin film, set the vacuum pressure to 40 mbar for 15 min, then purge with nitrogen gas for 30 min to ensure removal of residue solvent.</li>
	<li>Hydrate the thin film using 1 mL of phosphate-buffered saline (PBS, pH 7.4) and seal with parafilm before agitating the flask using a rotary evaporator over a 60 &#176;C water bath for 20 min in order to form vesicles.</li>
	<li>Subject the hydrated suspension to 7 cycles of freeze and thaw by freezing in liquid N2 for 3 min followed by immersion in a 45 &#176;C water bath for 7 min.</li>
	<li>Extrude liposomes through a membrane using a mini-extruder (see Table of Materials) and 1.0 &#181;m track-etched polycarbonate membranes (see Table of Materials) for 2-6 cycles in order to obtain large unilamellar vesicles. 
	NOTE: In order to achieve more favorable targeting towards macrophages, manipulate extrusion cycles until the diameter of liposomes is approximately 1 &#181;m26,27,28.</li>
	<li>Condense the fresh liposomes into a pellet by ultracentrifugation at 186,000 x g and incubate with 1 mL of 1% poloxamer 188 (see Table of Materials) solution (10 mg/mL in PBS) at 45 &#176;C and 750 rpm for 30 min using a thermomixer with a 2 mL microcentrifuge tube attachment (see Table of Materials).</li>
	<li>Ultracentrifuge the mixture at 186,000 x g for 1 h at 4 &#176;C and remove the supernatant in order to remove any unbound polymer or drug. Store the liposomes pellets at 4 &#176;C, protected from light.</li>
</ol>
2. Characterization of Liposome Size and Zeta Potential
<ol>
	<li>Dilute liposome formulation with ultrapure water (1:100) and measure the particle size, polydispersity index (PDI) and zeta potential of the liposomes using a dynamic light scattering analyzer (see Table of Materials), in triplicate at 25 &#176;C. 
	NOTE: The PDI is a measure of the size distribution of the nanoparticle population. PDI values &#60; 0.05 indicates a more uniform distribution while values closer to 1 indicate a larger size distribution. The zeta potential is the overall surface charge.</li>
</ol>
3. Calculation of Entrapment Efficiency and Drug Loading in Liposomes
NOTE: To determine the entrapment efficiency of drug in liposomes, the drug content contained in the supernatant and liposome pellet are measured.
<ol>
	<li>Resuspend the liposome pellet in 1 mL of PBS.</li>
	<li>Remove the supernatant, dilute 10 x with ultrapure water and analyze by high-performance liquid chromatography (HPLC).</li>
	<li>To determine the drug concentration within the liposomes, add 100 &#181;L of liposome suspension to 900 &#181;L of 10% triton-X100 solution (to destroy the lipid membrane, releasing the entrapped drug and preventing lipid build up within the HPLC column) and vortex for 3 min before analysis.</li>
	<li>Inject 20 &#956;L of sample into an HPLC system consisting of a quaternary pump, thermostat auto-sampler and a 3 &#956;m 250 x 4.6 mm column. Set the mobile phase (10 mM PBS pH 2.5, acetonitrile and methanol (30:40:30, v/v/v)) flow rate to 0.9 mL/min and obtain spectral profiles using a diode array detector set at 230 mM.</li>
	<li>Calculate the entrapment efficiency (EE) and drug loading (DL) using the following equations: 
	EE (%) = [Me / Mt] x 100 
	DL (%) = [Me / Mlip] x 100 
	Where Me is the mass of drug encapsulated, Mt is the total mass of drug used during formulation and Mlip is the total mass of the lipids (including cholesterol) and encapsulated drug.</li>
</ol>
4. Preparation and Injection of Liposomes
<ol>
	<li>Prepare borosilicate microinjection needles by inserting a thin wall borosilicate glass capillary (1 mm O.D. x 0.78 mm I.D. x 10 cm length) into a micropipette puller device (see Table of Materials) with the following generic puller settings (heat 680, pull 75, velocity 40, time 55 and pressure 530) to produce a tapered microinjection needle.</li>
	<li>Place the needles into a Petri dish onto the modeling clay and arrange in a way that the pulled end is not touching the bottom of the dish. Keep the Petri dish lid closed to avoid dust contamination until ready for use.</li>
	<li>Dilute freshly-prepared liposome formulations 1:1 in sterile PBS (pH 7.4) and briefly vortex (2-3 s) to mix. 
	NOTE: Protect liposomes from light until larvae are ready for microinjection.</li>
	<li>Set up adult zebrafish breeding pairs the night before and collect embryos as previously described by Rosen et al.29.</li>
	<li>Transfer the embryos into a Petri dish (100 mm x 20 mm style with approximately 60 embryos per dish) containing E3 medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4) supplemented with 0.1% methylene blue to inhibit fungal growth. Remove all dead or unfertilized embryos and incubate overnight at 28.5 &#176;C.</li>
	<li>To facilitate live imaging, add 0.003% N-Phenylthiourea (PTU) to the E3 media when the embryos reach 24 h of development to prevent pigment formation (melanization).</li>
	<li>Manually dechorionate the embryos at approximately 30 h post fertilization (hpf) using fine tip forceps. 
	NOTE: Do not use enzymatic treatment to dechorionate embryos as this can cause epithelial damage and local inflammation thereby influencing immunological studies</li>
	<li>Let the embryos develop at 28.5 &#176;C until 2 days post fertilization (dpf).</li>
	<li>Prepare an injection plate by pouring enough 3% methyl cellulose (in E3 media) into the lid of a small 35 mm culture dish to form a thin film covering the entire surface. 
	NOTE: When preparing 3% methyl cellulose in E3 media, it takes several days for the methyl cellulose to dissolve with gentle agitation on an orbital shaker.</li>
	<li>Anaesthetize the larvae by incubating in E3 media supplemented with 200 &#181;g/mL of Tricaine.</li>
	<li>Collect a pool of approximately 20-25 larvae using a plastic transfer pipette and allow the embryos to concentrate at the tip of the pipette by gravity. Taking care to transfer the larvae with minimal liquid, place them in the middle of the 35 mm culture dish lid.</li>
	<li>Using a microcapillary loader, arrange the larvae in the following ways: anterior towards the top of the injection plate with the dorsal surface facing toward the microinjection needle, to inject into the hindbrain ventricle (Figure 2A-D); or, anterior towards the left of the injection plate (when injecting from the right) with the ventral surface facing towards the microinjection needle to inject into the sinus venosus (Figure 2I). 
	NOTE: When injected this way, hindbrain ventricle-resident (Figure 2E) and caudal hematopoietic tissue(CHT)-resident (Figure 2J) macrophages can be targeted, respectively. The CHT region of larval zebrafish is a hematopoietic site analogous to the mammalian fetal liver. Pay extra attention not to damage the embryos while arranging as this may cause local inflammation thereby influencing immunological studies.</li>
	<li>Take freshly prepared liposomes (from step 4.3) and back fill an injection needle with approximately 5 &#956;L of the liposome injection mix using an microcapillary loader (see Table of Materials). 
	NOTE: When assessing the localization of liposomes to the phagolysosomal compartments of macrophages (Figure 2H), supplement this injection mix with 200 &#956;g/mL of a red fluorescent marker of phagosomes (see Table of Materials) and 100 nM of a far red fluorescent marker of lysosomes (see Table of Materials) to mark phagosomes30,31 and lysosomes32, respectively.</li>
	<li>Mount the needle into a micromanipulator (see Table of Materials) connected to a magnetic stand and a pressure injector (see Table of Materials). Position under a stereomicroscope and set the injector to a pulse duration of approximately 50 ms and an injection pressure of approximately 40 psi.</li>
	<li>Cut the tip of the microinjection needle with clean fine tip forceps to generate an opening of approximately 5 &#956;m in diameter.</li>
	<li>Calibrate the volume to be injected by injecting a bolus into a drop of mineral oil positioned over the grid lines of a hemocytometer. Adjust the pulse duration and/or the injection pressure until the diameter of the bolus covers 2.5 of the smallest grid lines (this corresponds to a volume of approximately 1 nL).</li>
	<li>Carefully inject 1 nL of the liposome injection mix into the hindbrain ventricle of each larvae. 
	NOTE: When injecting into the hindbrain ventricle, take care not to penetrate too far ventrally (beyond the hindbrain) as this can disrupt the underlying vasculature leading to hemorrhages. When injecting into the sinus venosus, care should be taken to ensure the tip of the needle is just within the pericardium and not penetrating the yolk.</li>
	<li>Following injection, immediately transfer the injected larvae back into E3 media by adding E3 media to the injection plate and gently agitating the larvae out of the methyl cellulose using a plastic transfer pipette.</li>
	<li>Incubate the larvae at 28.5 &#176;C (protected from light) until analysis by live confocal imaging.</li>
</ol>
5. Confocal Imaging and Image Analysis to Confirm Macrophage Targeting of Liposomes
<ol>
	<li>Heat a water bath to 50 &#176;C.</li>
	<li>Anaesthetize liposome-injected larvae by transferring to a new Perti dish containing E3 media supplemented with 0.003% PTU and 200 &#181;g/mL Tricaine.</li>
	<li>Prepare live imaging mounting medium by dissolving 1% low melting point agarose in E3 media, supplemented with 0.003% PTU, in a microwave. Place in the water bath and once the solution has cooled to 50 &#176;C, add 200 &#181;g/mL of Tricaine.</li>
	<li>Keep the mounting medium in the 50 &#176;C water bath to prevent premature polymerization of the agarose.</li>
	<li>To image the dorsal surface of the hindbrain on a confocal microscope equipped with a water immersion lens (Figure 2E), embed the larvae as follows: Fill a 35 mm culture dish with the mounting medium (to a depth of approximately 5 mm) and let it polymerize. Excavate a small trench in the agarose that is of sufficient size to accommodate the yolk sac.</li>
	<li>Fill a plastic transfer pipette with molten mounting medium, expel a small quantity, and use it to collect the anaesthetized larvae. Immediately transfer the larvae into the culture dish and orientate the yolk sacs, ventral side down, into the excavated holes, using a microcapillary loader. Periodically maintain them in this position until the agarose polymerizes. Overlay with E3 media supplemented with 200 &#181;g/mL Tricaine. 
	NOTE: When transferring and positioning larvae, take care not to cause epithelial damage and local inflammation which can influence immunological studies. For positioning of larvae to live image the CHT region (Figure 2J), mount as described above, but position the larvae within the excavated hole laterally.</li>
	<li>When imaging on an inverted confocal microscope, embed the larvae, without the agarose bed, directly on the bottom of a glass bottom dish. Arrange them dorsal surface down (or laterally) and following polymerization, cover the entire dish surface with an even layer of mounting medium. Once polymerized, add a thin layer of E3 media supplemented with 200 &#181;g/mL of Tricaine.</li>
	<li>To live image the hindbrain ventricle on a confocal microscope, use the following settings: 512 x 512 pixels; 40 x 3 &#956;m Z-stacks (extending from dorsal-most surface of the hindbrain); 20x objective; 2.5x zoom. 
	NOTE: When comparing signal intensities between samples (for example when imaging the uptake of Marina Blue-labeled liposomes injected within reporter lines marking either macrophages [Tg(mpeg1:EGFP)33] or neutrophils [Tg(lyz:EGFP)34]), it is essential that laser settings are kept the same (e.g., pinhole diameter, laser voltage, offset and gain) to help ensure any differences are not an artifact of altered image acquisition settings.</li>
	<li>Use 3D image analysis software to quantify macrophage or neutrophil uptake of Marina Blue-labeled liposomes. To quantify the number of cells containing liposomes (Figure 2F) within a Z-stack, scroll through the individual Z sections and count the number of individual cells containing Marina Blue-labeled liposomes. To quantify the fluorescence intensity of liposomes within cells (Figure 2G), draw a region of interest around each cell (in the X, Y and Z dimensions) to quantify the total or mean fluorescence intensity of intracellular Marina Blue.</li>
</ol>

<ol>
	<li>Chow, A., Brown, B. D., Merad, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+the+mononuclear+phagocyte+system+in+the+molecular+age.">Studying the mononuclear phagocyte system in the molecular age.</a> Nature Reviews Immunology. 11 (11), 788-798 (2011).</li><li>Li, Q., Barres, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia+and+macrophages+in+brain+homeostasis+and+disease.">Microglia and macrophages in brain homeostasis and disease.</a> Nature Reviews Immunology. 18 (4), 225-242 (2018).</li><li>Krenkel, O., Tacke, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+macrophages+in+tissue+homeostasis+and+disease.">Liver macrophages in tissue homeostasis and disease.</a> Nature Reviews Immunology. 17 (5), 306-321 (2017).</li><li>Alderton, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumour+immunology:+turning+macrophages+on,+off+and+on+again.">Tumour immunology: turning macrophages on, off and on again.</a> Nature Reviews Immunology. 14 (3), 136-137 (2014).</li><li>Moore, K. J., Sheedy, F. J., Fisher, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophages+in+atherosclerosis:+a+dynamic+balance.">Macrophages in atherosclerosis: a dynamic balance.</a> Nature Reviews Immunology. 13 (10), 709-721 (2013).</li><li>Lawrence, T., Natoli, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+regulation+of+macrophage+polarization:+enabling+diversity+with+identity.">Transcriptional regulation of macrophage polarization: enabling diversity with identity.</a> Nature Reviews Immunology. 11 (11), 750-761 (2011).</li><li>Chawla, A., Nguyen, K. D., Goh, Y. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage-mediated+inflammation+in+metabolic+disease.">Macrophage-mediated inflammation in metabolic disease.</a> Nature Reviews Immunology. 11 (11), 738-749 (2011).</li><li>Renshaw, S. A., Trede, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+model+450+million+years+in+the+making:+zebrafish+and+vertebrate+immunity.">A model 450 million years in the making: zebrafish and vertebrate immunity.</a> Disease models and mechanisms. 5 (1), 38-47 (2012).</li><li>Hall, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunoresponsive+gene+1+augments+bactericidal+activity+of+macrophage-lineage+cells+by+regulating+beta-oxidation-dependent+mitochondrial+ROS+production.">Immunoresponsive gene 1 augments bactericidal activity of macrophage-lineage cells by regulating beta-oxidation-dependent mitochondrial ROS production.</a> Cell Metabolism. 18 (2), 265-278 (2013).</li><li>Hall, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blocking+fatty+acid-fueled+mROS+production+within+macrophages+alleviates+acute+gouty+inflammation.">Blocking fatty acid-fueled mROS production within macrophages alleviates acute gouty inflammation.</a> Journal of Clinical Investigation. 125 (5), 1752-1771 (2018).</li><li>Cambier, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mycobacteria+manipulate+macrophage+recruitment+through+coordinated+use+of+membrane+lipids.">Mycobacteria manipulate macrophage recruitment through coordinated use of membrane lipids.</a> Nature. 505 (7482), 218-222 (2014).</li><li>Davis, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+visualization+of+mycobacterium-macrophage+interactions+leading+to+initiation+of+granuloma+formation+in+zebrafish+embryos.">Real-time visualization of mycobacterium-macrophage interactions leading to initiation of granuloma formation in zebrafish embryos.</a> Immunity. 17 (6), 693-702 (2002).</li><li>Madigan, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Macrophage+Response+to+Mycobacterium+leprae+Phenolic+Glycolipid+Initiates+Nerve+Damage+in+Leprosy.">A Macrophage Response to Mycobacterium leprae Phenolic Glycolipid Initiates Nerve Damage in Leprosy.</a> Cell. 170 (5), 973-985 (2017).</li><li>Tobin, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+lta4h+locus+modulates+susceptibility+to+mycobacterial+infection+in+zebrafish+and+humans.">The lta4h locus modulates susceptibility to mycobacterial infection in zebrafish and humans.</a> Cell. 140 (5), 717-730 (2010).</li><li>Volkman, H. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tuberculous+granuloma+induction+via+interaction+of+a+bacterial+secreted+protein+with+host+epithelium.">Tuberculous granuloma induction via interaction of a bacterial secreted protein with host epithelium.</a> Science. 327 (5964), 466-469 (2010).</li><li>Bowman, T. V., Zon, L. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Swimming+into+the+future+of+drug+discovery:+in+vivo+chemical+screens+in+zebrafish.">Swimming into the future of drug discovery: in vivo chemical screens in zebrafish.</a> ACS Chemical Biology. 5 (2), 159-161 (2010).</li><li>Kaufman, C. K., White, R. M., Zon, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+genetic+screening+in+the+zebrafish+embryo.">Chemical genetic screening in the zebrafish embryo.</a> Nature Protocols. 4 (10), 1422-1432 (2009).</li><li>Zon, L. I., Peterson, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+drug+discovery+in+the+zebrafish.">In vivo drug discovery in the zebrafish.</a> Nature Reviews Drug Discovery. 4 (1), 35-44 (2005).</li><li>Malam, Y., Loizidou, M., Seifalian, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liposomes+and+nanoparticles:+nanosized+vehicles+for+drug+delivery+in+cancer.">Liposomes and nanoparticles: nanosized vehicles for drug delivery in cancer.</a> Trends in Pharmacological Sciences. 30 (11), 592-599 (2009).</li><li>Torchilin, V. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+with+liposomes+as+pharmaceutical+carriers.">Recent advances with liposomes as pharmaceutical carriers.</a> Nature Reviews Drug Discovery. 4 (2), 145-160 (2005).</li><li>Immordino, M. L., Dosio, F., Cattel, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stealth+liposomes:+review+of+the+basic+science,+rationale,+and+clinical+applications,+existing+and+potential.">Stealth liposomes: review of the basic science, rationale, and clinical applications, existing and potential.</a> International Journal of Nanomedicine. 1 (3), 297-315 (2006).</li><li>Astin, J. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innate+immune+cells+and+bacterial+infection+in+zebrafish.">Innate immune cells and bacterial infection in zebrafish.</a> Methods in Cell Biology. 138, 31-60 (2017).</li><li>Zhang, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post-insertion+of+poloxamer+188+strengthened+liposomal+membrane+and+reduced+drug+irritancy+and+in+vivo+precipitation,+superior+to+PEGylation.">Post-insertion of poloxamer 188 strengthened liposomal membrane and reduced drug irritancy and in vivo precipitation, superior to PEGylation.</a> Journal of Controlled Release. 203, 161-169 (2015).</li><li>Wu, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liposome-Mediated+Drug+Delivery+in+Larval+Zebrafish+to+Manipulate+Macrophage+Function.">Liposome-Mediated Drug Delivery in Larval Zebrafish to Manipulate Macrophage Function.</a> Zebrafish. 16 (2), 171-181 (2019).</li><li>Cader, M. Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C13orf31+(FAMIN)+is+a+central+regulator+of+immunometabolic+function.">C13orf31 (FAMIN) is a central regulator of immunometabolic function.</a> Nature Immunology. 17 (9), 1046-1056 (2016).</li><li>Chono, S., Tanino, T., Seki, T., Morimoto, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+particle+size+on+drug+delivery+to+rat+alveolar+macrophages+following+pulmonary+administration+of+ciprofloxacin+incorporated+into+liposomes.">Influence of particle size on drug delivery to rat alveolar macrophages following pulmonary administration of ciprofloxacin incorporated into liposomes.</a> Journal of Drug Targeting. 14 (8), 557-566 (2006).</li><li>Chono, S., Tanino, T., Seki, T., Morimoto, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uptake+characteristics+of+liposomes+by+rat+alveolar+macrophages:+influence+of+particle+size+and+surface+mannose+modification.">Uptake characteristics of liposomes by rat alveolar macrophages: influence of particle size and surface mannose modification.</a> Journal of Pharmact and Pharmacology. 59 (1), 75-80 (2007).</li><li>Chono, S., Tauchi, Y., Morimoto, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+particle+size+on+the+distributions+of+liposomes+to+atherosclerotic+lesions+in+mice.">Influence of particle size on the distributions of liposomes to atherosclerotic lesions in mice.</a> Drug Development and Industrial Pharmacy. 32 (1), 125-135 (2006).</li><li>Rosen, J. N., Sweeney, M. F., Mably, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+zebrafish+embryos+to+analyze+gene+function.">Microinjection of zebrafish embryos to analyze gene function.</a> Journal of Visualized Experiments. (25), (2009).</li><li>Hall, C., Flores, M. V., Crosier, K., Crosier, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+cell+imaging+of+zebrafish+leukocytes.">Live cell imaging of zebrafish leukocytes.</a> Methods in Molecular Biology. 546, 255-271 (2009).</li><li>Kapellos, T. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+real+time+imaging+platform+to+quantify+macrophage+phagocytosis.">A novel real time imaging platform to quantify macrophage phagocytosis.</a> Biochemical Pharmacology. 116, 107-119 (2016).</li><li>Shen, K., Sidik, H., Talbot, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Rag-Ragulator+Complex+Regulates+Lysosome+Function+and+Phagocytic+Flux+in+Microglia.">The Rag-Ragulator Complex Regulates Lysosome Function and Phagocytic Flux in Microglia.</a> Cell Reports. 14 (3), 547-559 (2016).</li><li>Ellett, F., Pase, L., Hayman, J. W., Andrianopoulos, A., Lieschke, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mpeg1+promoter+transgenes+direct+macrophage-lineage+expression+in+zebrafish.">mpeg1 promoter transgenes direct macrophage-lineage expression in zebrafish.</a> Blood. 117 (4), 49-56 (2011).</li><li>Hall, C., Flores, M. V., Storm, T., Crosier, K., Crosier, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+lysozyme+C+promoter+drives+myeloid-specific+expression+in+transgenic+fish.">The zebrafish lysozyme C promoter drives myeloid-specific expression in transgenic fish.</a> BMC Developmental Biology. 7, 42 (2007).</li><li>Ahsan, F., Rivas, I. P., Khan, M. A., Torres Suarez, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+to+macrophages:+role+of+physicochemical+properties+of+particulate+carriers--liposomes+and+microspheres--on+the+phagocytosis+by+macrophages.">Targeting to macrophages: role of physicochemical properties of particulate carriers--liposomes and microspheres--on the phagocytosis by macrophages.</a> Journal of Controlled Release. 79 (1-3), 29-40 (2002).</li><li>Martin, W. J., Walton, M., Harper, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resident+macrophages+initiating+and+driving+inflammation+in+a+monosodium+urate+monohydrate+crystal-induced+murine+peritoneal+model+of+acute+gout.">Resident macrophages initiating and driving inflammation in a monosodium urate monohydrate crystal-induced murine peritoneal model of acute gout.</a> Arthritis and Rheumatology. 60 (1), 281-289 (2009).</li><li>Faires, J. S., McCarty, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+arthritis+in+man+and+dog+after+intrasynovial+injection+of+sodium+urate+crystals.">Acute arthritis in man and dog after intrasynovial injection of sodium urate crystals.</a> Lancet. 280, 682-685 (1962).</li><li>Martin, W. J., Harper, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innate+inflammation+and+resolution+in+acute+gout.">Innate inflammation and resolution in acute gout.</a> Immunology and Cell Biology. 88 (1), 15-19 (2010).</li><li>Fenaroli, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoparticles+as+drug+delivery+system+against+tuberculosis+in+zebrafish+embryos:+direct+visualization+and+treatment.">Nanoparticles as drug delivery system against tuberculosis in zebrafish embryos: direct visualization and treatment.</a> ACS Nano. 8 (7), 7014-7026 (2014).</li><li>Robertson, J. D., Ward, J. R., Avila-Olias, M., Battaglia, G., Renshaw, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+Neutrophilic+Inflammation+Using+Polymersome-Mediated+Cellular+Delivery.">Targeting Neutrophilic Inflammation Using Polymersome-Mediated Cellular Delivery.</a> Journal of Immunology. 198 (9), 3596-3604 (2017).</li><li>Le Guellec, D., Morvan-Dubois, G., Sire, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin+development+in+bony+fish+with+particular+emphasis+on+collagen+deposition+in+the+dermis+of+the+zebrafish+(Danio+rerio).">Skin development in bony fish with particular emphasis on collagen deposition in the dermis of the zebrafish (Danio rerio).</a> International Journal of Developmental Biology. 48 (2-3), 217-231 (2004).</li><li>Kelly, C., Jefferies, C., Cryan, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+liposomal+drug+delivery+to+monocytes+and+macrophages.">Targeted liposomal drug delivery to monocytes and macrophages.</a> Journal of Drug Delivery. 2011, 727241 (2011).</li><li>Fidler, I. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+of+liposomes+to+improve+delivery+of+macrophage-augmenting+agents+to+alveolar+macrophages.">Design of liposomes to improve delivery of macrophage-augmenting agents to alveolar macrophages.</a> Cancer Research. 40 (12), 4460-4466 (1980).</li><li>Ng, A. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+the+digestive+system+in+zebrafish:+III.+Intestinal+epithelium+morphogenesis.">Formation of the digestive system in zebrafish: III. Intestinal epithelium morphogenesis.</a> Developmenal Biology. 286 (1), 114-135 (2005).</li></ol>

The authors declare that no competing financial interests exist.

Here, we have provided a detailed protocol to formulate drug-loaded liposomes to specifically target macrophages in larval zebrafish. This method can be used to dissect the role of macrophages in certain disease models by ensuring direct targeted delivery of drugs specifically to macrophages. Moreover, it can be used when general toxicity of drugs limits their use when delivered by more conventional routes, like immersion. The protocol described here provides an alternative to other nanoparticulate systems that have been...

The mononuclear phagocyte system provides a first line of defense against invading pathogens. This system consists of monocytes, monocyte-derived dendritic cells and macrophages, which actively phagocytoze foreign pathogens, thereby limiting pathogen spread. In addition to these phagocytic and microbicidal effector functions, dendritic cells and macrophages are also capable of cytokine production and antigen-presentation to activate the adaptive immune system1. Of these cells, macrophages have received particular attention due to their diverse functional heterogeneity and involvement in multiple inflammatory diseases, from autoimmunity and infectious diseases to cancer2,3,4,5,6,7. The plasticity of macrophages and their ability to functionally adapt to the needs to their tissue environment necessitates experimental approaches to directly observe and interrogate these cells in vivo.
Larval zebrafish are an ideal model organism by which to study the function and plasticity of macrophages in vivo8. The optical transparency of larval zebrafish provides a window to directly observe the behavior of macrophages, especially when coupled with macrophage-marking transgenic reporter lines. Exploiting the live imaging potential and experimental tractability of larval zebrafish has led to many significant insights into macrophage function that have direct relevance to human disease9,10,11,12,13,14,15. Many of these studies have also taken advantage of the high conservation of drug activity in zebrafish (an attribute that underpins their use as a whole animal drug discovery platform16,17,18), by utilizing chemical interventions to pharmacologically manipulate macrophage function. To date, these pharmacological treatments have been mostly delivered either through immersion, which requires the drug to be water soluble, or by direct microinjection of free drug (Figure 1A). Limitations of these passive delivery strategies include off-target effects and general toxicity that may preclude assessing any impact on macrophage function. Additionally, when investigating drug effects on macrophages it is unknown whether the drugs are acting on the macrophages themselves or through more indirect mechanisms. When performing similar chemical intervention studies to investigate macrophage function, we recognized there was an unmet need to develop an inexpensive and straightforward delivery method to target drugs specifically to macrophages.
Liposomes are microscopic, biocompatible, lipid bilayered vesicles that can encapsulate proteins, nucleotides and drug cargo19. The unilamellar or multilamellar lipid bilayer structure of liposomes forms an aqueous inner lumen where water-soluble drugs can be incorporated while hydrophobic drugs can be integrated into the lipid membranes. In addition, the physicochemical properties of liposomes, including size, charge and surface modifications can be manipulated to tailor their targeting to specific cells20,21. These features of liposomes have made them an attractive vehicle to deliver drugs and enhance the precision of current treatment regimens20. As liposomes are naturally phagocytozed by macrophages (a feature exploited by their routine use in delivering clodronate specifically to macrophages for ablation experiments22), they present as an attractive option for macrophage-specific drug delivery (Figure 1B).
This protocol describes the formulation of drugs into blue fluorescent liposomes coated with the hydrophilic polymer poloxamer 188, that forms a protective layer on the liposome surface and has been shown to enhance drug retention and have superior biocompatibility23. Poloxamer was chosen for surface coating of liposomes as our previous research had shown that, when compared to polyethylene glycol modified liposomes, poloxamer modified liposomes showed better biocompatibility following subcutaneous injection of rat paws and similar pharmacokinetics in rabbits following intravenous infusion23. Protocols are also described for their microinjection into larval zebrafish and live imaging to assess their macrophage-targeting ability and localization to intracellular compartments necessary for liposome degradation and cytoplasmic drug delivery. As a proof-of-concept we have previously used this technique to target two drugs to macrophages to suppress their activation in a larval zebrafish model of acute gouty inflammation24. This drug delivery technique expands the chemical &#34;toolkit&#34; available to zebrafish researchers wanting to ensure macrophage-targeting of their drugs of interest.

<table><tbody><tr><td>1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)</td><td>Avanti Polar Lipids, Inc.</td><td>850355P</td><td></td></tr><tr><td>1,2-diseteroyl-sn-glycero-3-phosphocholine (DSPE)</td><td>Avanti Polar Lipids, Inc.</td><td>850367P</td><td></td></tr><tr><td>1.0 &amp;micro;m Whatman Nuclepore Track-Etched polycarbonate membranes</td><td>GE Healthcare Life Sciences</td><td>110610</td><td></td></tr><tr><td>25 mL round-bottom flask</td><td>Sigma-Aldrich</td><td>Z278262</td><td></td></tr><tr><td>35 mm culture dish</td><td>Thermo Scientific</td><td>150460</td><td></td></tr><tr><td>Acetonitrile</td><td>Sigma-Aldrich</td><td>34998</td><td></td></tr><tr><td>Agilent 1260 Infinity Diode Array Detector</td><td>Agilent Technologies</td><td>G4212B</td><td></td></tr><tr><td>Agilent 1260 Infinity Quaternary Pump</td><td>Agilent Technologies</td><td>G1311B</td><td></td></tr><tr><td>Agilent 1290 Infinity Series Thermostat</td><td>Agilent Technologies</td><td>G1330B</td><td></td></tr><tr><td>Avanti mini-extruder Avanti Polar Lipids Inc.</td><td>Avanti Polar Lipids Inc.</td><td></td><td></td></tr><tr><td>borosilicate microinjection needles</td><td>Warner Instruments</td><td>203-776-0664</td><td></td></tr><tr><td>CaCl2</td><td>Sigma-Aldrich</td><td>C4901-100G</td><td></td></tr><tr><td>cholesterol</td><td>Sigma-Aldrich</td><td>C8667</td><td></td></tr><tr><td>Dumont No.5 fine tip forceps</td><td>Fine Science Tools</td><td>11251-10</td><td></td></tr><tr><td>Eppendorf Microloader pipette tip</td><td>Eppendorf</td><td>5242956003</td><td></td></tr><tr><td>Eppendorf SmartBlock 1.5 mL, thermoblock for 24 reaction vessels</td><td>Eppendorf</td><td>4053-6038</td><td></td></tr><tr><td>eyelash manipulator</td><td>Ted Pella Inc.</td><td>113</td><td></td></tr><tr><td>hemocytometer</td><td>Hawksley</td><td>BS.748</td><td></td></tr><tr><td>HEPES</td><td>BDH Chemicals</td><td>441474J</td><td></td></tr><tr><td>HPLC system</td><td>Agilent Technologies</td><td>1260 series HPLC system</td><td></td></tr><tr><td>KCl</td><td>Sigma-Aldrich</td><td>P9541-1KG</td><td></td></tr><tr><td>low melting point agarose</td><td>Invitrogen</td><td>16520-100</td><td></td></tr><tr><td>LysoTracker Deep Red</td><td>Invitrogen</td><td>L12492</td><td>1 mM stock solution in DMSO, keep at -20 &amp;deg;C and protect from light.</td></tr><tr><td>LysoTracker Deep Red</td><td>Thermo Scientific</td><td>L12492</td><td></td></tr><tr><td>magnetic stand</td><td>Narishige</td><td>GJ-1</td><td></td></tr><tr><td>Marina Blue 1,2-dihexadecanoyl-sn-glycero-phosphoethanolamine (Marina Blue DHPE)</td><td>Invitrogen</td><td>M12652</td><td>Keep at -20 &amp;deg;C and protect from light.</td></tr><tr><td>Methanol</td><td>Sigma-Aldrich</td><td>34860</td><td></td></tr><tr><td>methyl cellulose</td><td>Sigma-Aldrich</td><td>M0387-500G</td><td></td></tr><tr><td>methylene blue</td><td>Alfa Aesar</td><td>42771</td><td></td></tr><tr><td>MgSO4</td><td>Sigma-Aldrich</td><td>230391-500G</td><td></td></tr><tr><td>micromanipulator</td><td>Narishige</td><td>M-152</td><td></td></tr><tr><td>mineral oil</td><td>Sigma-Aldrich</td><td>M-3516</td><td></td></tr><tr><td>Mitochondria-targeting antioxidant MitoTEMPO</td><td>Sigma-Aldrich</td><td>SML0737</td><td></td></tr><tr><td>MitoSOX Red Mitochondrial Superoxide Indicator</td><td>Thermo Scientific</td><td>M36008</td><td></td></tr><tr><td>MitoTEMPO</td><td>Sigma-Aldrich</td><td>SML0737</td><td>Keep at -20 &amp;deg;C and protect from light.</td></tr><tr><td>N-Phenylthiourea (PTU)</td><td>Sigma-Aldrich</td><td>P7629-10G</td><td>Take care when handling, toxic.</td></tr><tr><td>NaCl</td><td>BDH Chemicals</td><td>27810.295</td><td></td></tr><tr><td>PBS (pH 7.4)</td><td>Gibco</td><td>10010-023</td><td></td></tr><tr><td>Petri dish (100 mm x 20 mm)</td><td>Corning Inc.</td><td>430167</td><td></td></tr><tr><td>Phenomenex C18 Gemini-NZ 3 mm 250 mm x 4.6 mm column</td><td>Phenomenex</td><td>00G-4439-E0</td><td></td></tr><tr><td>pHrodo Red Escherichia coli BioParticles Conjugate</td><td>Thermo Scientific</td><td>P35361</td><td></td></tr><tr><td>pHrodo Red Escherichia coli BioParticles Conjugate</td><td>Invitrogen</td><td>P35361</td><td>Keep at -20 &amp;deg;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.</td></tr><tr><td>plastic transfer pipette</td><td>Medi&#039;Ray</td><td>RL200C</td><td></td></tr><tr><td>poloxamer 188</td><td>BASF Corporation</td><td></td><td></td></tr><tr><td>pressure injector</td><td>Applied Scientific Instruments</td><td>MPPI-2</td><td></td></tr><tr><td>rotary evaporator</td><td>B&amp;uuml;chi, Flawil, Switzerland</td><td>B&amp;uuml;chi R-215 Rotavapor</td><td></td></tr><tr><td>Scanning confocal microscope</td><td>Olympus</td><td>Olympus FV1000 FluoView</td><td></td></tr><tr><td>Sorvall WX+ Ultracentrifuge</td><td>Thermo Scientific</td><td>75000090</td><td></td></tr><tr><td>stereomicroscope</td><td>Leica</td><td>MZ12</td><td></td></tr><tr><td>Tricaine</td><td>Sigma-Aldrich</td><td>A5040-25G</td><td>Make 4 mg/mL stock solution (in deionzed H2O) and keep at -20 &amp;deg;C.</td></tr><tr><td>triton-X100</td><td>Sigma-Aldrich</td><td>X100-100ML</td><td></td></tr><tr><td>Ultrasonic bath</td><td>Thermo Scientific</td><td>FB-11205</td><td></td></tr><tr><td>Volocity Image Analysis Software</td><td>PerkinElmer</td><td>version 6.3</td><td></td></tr><tr><td>water bath</td><td></td><td></td><td></td></tr><tr><td>Zetasizer Nano</td><td>Malvern Instruments Ltd</td><td>Zetasizer Nano ZS ZEN 3600</td><td></td></tr></tbody></table>

targeting drugs to larval zebrafish macrophages by injecting drug-loaded liposomes

Zebrafish (Danio rerio) larvae have developed into a popular model to investigate host-pathogen interactions and the contribution of innate immune cells to inflammatory disease due to their functionally conserved innate immune system. They are also widely used to examine how innate immune cells help guide developmental processes. By taking advantage of the optical transparency and genetic tractability of larval zebrafish, these studies often focus on live imaging approaches to functionally characterize fluorescently marked macrophages and neutrophils within intact animals. Due to their diverse functional heterogeneity and ever-expanding roles in disease pathogenesis, macrophages have received significant attention. In addition to genetic manipulations, chemical interventions are now routinely used to manipulate and examine macrophage behavior in larval zebrafish. Delivery of these drugs is typically limited to passive targeting of free drug through direct immersion or microinjection. These approaches rely on the assumption that any changes to macrophage behavior are the result of a direct effect of the drug on the macrophages themselves, and not a downstream consequence of a direct effect on another cell type. Here, we present our protocols for targeting drugs specifically to larval zebrafish macrophages by microinjecting drug-loaded fluorescent liposomes. We reveal that poloxamer 188-modified drug-loaded blue fluorescent liposomes are readily taken up by macrophages, and not by neutrophils. We also provide evidence that drugs delivered in this way can impact macrophage activity in a manner consistent with the mechanism of action of the drug. This technique will be of value to researchers wanting to ensure targeting of drugs to macrophages and when drugs are too toxic to be delivered by traditional methods like immersion.

The thin film hydration approach described here for the preparation of fluorescent liposomes enclosing drugs is a simple and cost-effective method. With the protocol used in this study, the liposomes are expected to be unilamellar23,24. The size, zeta potential, drug loading and entrapment efficiency of the liposomes produced are summarized in Table 1. The particle size of the liposomes (before and after drug loading) are similar (Table 1...

Watch this Scientific Journal Video about Targeting Drugs to Larval Zebrafish Macrophages by Injecting Drug-Loaded Liposomes at JoVE.com

Targeting Drugs to Larval Zebrafish Macrophages by Injecting Drug-Loaded Liposomes

Here, we describe the synthesis of drug-loaded liposomes and their microinjection into larval zebrafish for the purpose of targeting drug delivery to macrophage-lineage cells.

Zebrafish (Danio rerio) larvae have developed into a popular model to investigate host-pathogen interactions and the contribution of innate immune cells ...

targeting-drugs-to-larval-zebrafish-macrophages-injecting-drug-loaded

Research

JoVE Journal

Immunology and Infection

7.6K Views.  University of Auckland. Dit protocol beschrijft een techniek voor het richten van geneesmiddelen van belang zijn op larve zebravis macrofagen te manipuleren en te evalueren macrofaag functie. Deze techniek heeft voordelen ten opzichte van traditionele drug delivery strategieën zoals onderdompeling door ervoor te zorgen dat de drugs specifiek zijn gericht op macrofagen door liposoom inkapseling. Wanneer toegepast op zebravismodellen van ziekte met een ontstekingscomponent, heeft deze techniek het potentieel om op te...

Video: Targeting Drugs to Larval Zebrafish Macrophages by Injecting Drug-Loaded Liposomes

Infection of Zebrafish Embryos with Intracellular Bacterial Pathogens

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen interactions 1. The major cell types of the innate immune system, macrophages and neutrophils, develop during the first days of embryogenesis prior to the maturation of lymphocytes that are required for adaptive immune responses. The ease of obtaining large numbers of embryos, their accessibility due to external development, the optical transparency of embryonic and larval stages, a wide range of genetic tools, extensive mutant resources and collections of transgenic reporter lines, all add to the versatility of the zebrafish model. Salmonella enterica serovar Typhimurium (S. typhimurium) and Mycobacterium marinum can reside intracellularly in macrophages and are frequently used to study host-pathogen interactions in zebrafish embryos. The infection processes of these two bacterial pathogens are interesting to compare because S. typhimurium infection is acute and lethal within one day, whereas M. marinum infection is chronic and can be imaged up to the larval stage 2, 3. The site of micro-injection of bacteria into the embryo (Figure 1) determines whether the infection will rapidly become systemic or will initially remain localized. A rapid systemic infection can be established by micro-injecting bacteria directly into the blood circulation via the caudal vein at the posterior blood island or via the Duct of Cuvier, a wide circulation channel on the yolk sac connecting the heart to the trunk vasculature. At 1 dpf, when embryos at this stage have phagocytically active macrophages but neutrophils have not yet matured, injecting into the blood island is preferred. For injections at 2-3 dpf, when embryos also have developed functional (myeloperoxidase-producing) neutrophils, the Duct of Cuvier is preferred as the injection site. To study directed migration of myeloid cells towards local infections, bacteria can be injected into the tail muscle, otic vesicle, or hindbrain ventricle 4-6. In addition, the notochord, a structure that appears to be normally inaccessible to myeloid cells, is highly susceptible to local infection 7. A useful alternative for high-throughput applications is the injection of bacteria into the yolk of embryos within the first hours after fertilization 8. Combining fluorescent bacteria and transgenic zebrafish lines with fluorescent macrophages or neutrophils creates ideal circumstances for multi-color imaging of host-pathogen interactions. This video article will describe detailed protocols for intravenous and local infection of zebrafish embryos with S. typhimurium or M. marinum bacteria and for subsequent fluorescence imaging of the interaction with cells of the innate immune system.

Transparent zebrafish embryos have proved useful model hosts to visualize and functionally study interactions between innate immune cells and intracellular bacterial pathogens, such as Salmonella typhimurium and Mycobacterium marinum. Micro-injection of bacteria and multi-color fluorescence imaging are essential techniques involved in the application of zebrafish embryo infection models.

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen ...

Non-invasive Imaging of Disseminated Candidiasis in Zebrafish Larvae

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 to 40% attributable mortality6. Systemic candidiasis is normally controlled by innate immunity, and individuals with genetic defects in innate immune cell components such as phagocyte NADPH oxidase are more susceptible to candidemia7-9. Very little is known about the dynamics of C. albicans interaction with innate immune cells in vivo. Extensive in vitro studies have established that outside of the host C. albicans germinates inside of macrophages, and is quickly destroyed by neutrophils10-14. In vitro studies, though useful, cannot recapitulate the complex in vivo environment, which includes time-dependent dynamics of cytokine levels, extracellular matrix attachments, and intercellular contacts10, 15-18. To probe the contribution of these factors in host-pathogen interaction, it is critical to find a model organism to visualize these aspects of infection non-invasively in a live intact host. 
The zebrafish larva offers a unique and versatile vertebrate host for the study of infection. For the first 30 days of development zebrafish larvae have only innate immune defenses2, 19-21, simplifying the study of diseases such as disseminated candidiasis that are highly dependent on innate immunity. The small size and transparency of zebrafish larvae enable imaging of infection dynamics at the cellular level for both host and pathogen. Transgenic larvae with fluorescing innate immune cells can be used to identify specific cells types involved in infection22-24. Modified anti-sense oligonucleotides (Morpholinos) can be used to knock down various immune components such as phagocyte NADPH oxidase and study the changes in response to fungal infection5. In addition to the ethical and practical advantages of using a small lower vertebrate, the zebrafish larvae offers the unique possibility to image the pitched battle between pathogen and host both intravitally and in color. 
The zebrafish has been used to model infection for a number of human pathogenic bacteria, and has been instrumental in major advances in our understanding of mycobacterial infection3, 25. However, only recently have much larger pathogens such as fungi been used to infect larva5, 23, 26, and to date there has not been a detailed visual description of the infection methodology. Here we present our techniques for hindbrain ventricle microinjection of prim25 zebrafish, including our modifications to previous protocols. Our findings using the larval zebrafish model for fungal infection diverge from in vitro studies and reinforce the need to examine the host-pathogen interaction in the complex environment of the host rather than the simplified system of the Petri dish5.

The rapid development, small size and transparency of zebrafish are tremendous advantages for the study of innate immune control of infection1-4. Here we demonstrate techniques for infecting zebrafish larvae using the fungal pathogen Candida albicans by microinjection, methodology recently used to implicate phagocyte NADPH oxidase activity in control of fungal dimorphism5.

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 ...

An Ectopic Chemokine Expression Model for Testing Macrophage Recruitment In Vivo

Zebrafish are widely used in basic and biomedical research. Many zebrafish transgenic lines are currently available to label various types of cells. Owing to the transparent embryonic body of zebrafish, it is convenient for us to study the effect of one chemokine on the behavior of a certain type of cells in vivo. Here we provided a workflow to investigate the function of a chemokine on macrophage migration in vivo. We constructed a tissue-specific overexpression plasmid to overexpress IL-34 and injected the plasmid into one-cell stage transgenic fish embryos whose macrophages were specifically labeled by a fluorescent protein. We then used whole mount fluorescent in situ hybridization and immunostaining to detect the pattern of the chemokine expression and the number or location of macrophages. The injected WT embryos were raised to generate a stable transgenic line. Finally, we used confocal live imaging to directly observe macrophage behavior in the stable transgenic fish to study the function of IL-34 on macrophages in vivo.

To test the effect of a chemokine on macrophage recruitment in vivo, the whole mount in situ hybridization was used to detect the ectopic expression of the chemokine, and immunostaining was used to label macrophages. Live imaging was used for real-time observation of macrophage migration.

Zebrafish are widely used in basic and biomedical research. Many zebrafish transgenic lines are currently available to label various types of cells. Owing ...

Developmental Biology

Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos

Zebrafish (Danio rerio) embryos are increasingly used as an infection model to study the function of the vertebrate innate immune system in host-pathogen interactions.&#160;The ease of obtaining large numbers of embryos, their accessibility due to external development, their optical transparency as well as the availability of a wide panoply of genetic/immunological tools and transgenic reporter line collections, contribute to the versatility of this model.&#160;In this respect, the present manuscript describes the use of zebrafish as an&#160;in vivo&#160;model system to investigate the chronology of Mycobacterium abscessus infection. This human pathogen can exist either as smooth (S) or rough (R) variants, depending on cell wall composition, and their respective virulence can be imaged and compared in zebrafish embryos and larvae. Micro-injection of either S or R fluorescent variants directly in the blood circulation via the caudal vein, leads to chronic or acute/lethal infections, respectively. This biological system allows high resolution visualization and analysis of the role of mycobacterial cording in promoting abscess formation. In addition, the use of fluorescent bacteria along with transgenic zebrafish lines harbouring fluorescent macrophages produces a unique opportunity for multi-color imaging of the host-pathogen interactions. This article describes detailed protocols for the preparation of homogenous M. abscessus inoculum and for intravenous injection of zebrafish embryos for subsequent fluorescence imaging of the interaction with macrophages. These techniques open the avenue to future investigations involving mutants defective in cord formation and are dedicated to understand how this impacts on M. abscessus pathogenicity in a whole vertebrate.

Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos

Optically transparent zebrafish embryos are widely used to study and visualize in real time the interactions between pathogenic microorganisms and the innate immune cells. Micro-injection of Mycobacterium abscessus, combined with fluorescence imaging, is used to scrutinize essential pathogenic features such as cord formation in zebrafish embryos.

Zebrafish (Danio rerio) embryos are increasingly used as an infection model to study the function of the vertebrate innate immune system in host-pathogen ...

Visualization of Macrophage Lytic Cell Death During Mycobacterial Infection in Zebrafish Embryos via Intravital Microscopy

Zebrafish is an excellent model organism for studying innate immune cell behavior due to its transparent nature and reliance solely on its innate immune system during early development. The Zebrafish Mycobacterium marinum (M. marinum) infection model has been well-established in studying host immune response against mycobacterial infection. It has been suggested that different macrophage cell death types will lead to the diverse outcomes of mycobacterial infection. Here we describe a protocol using intravital microscopy to observe macrophage cell death in zebrafish embryos following M. marinum infection. Zebrafish transgenic lines that specifically label macrophages and neutrophils are infected via intramuscular microinjection of fluorescently labeled M. marinum in either the midbrain or the trunk. Infected zebrafish embryos are subsequently mounted on low melting agarose and observed by confocal microscopy in X-Y-Z-T dimensions. Because long-term live imaging requires using low laser power to avoid photobleaching and phototoxicity, a strongly expressing transgenic is highly recommended. This protocol facilitates the visualization of the dynamic processes in vivo, including immune cell migration, host pathogen interaction, and cell death.

This protocol describes a technique for visualizing macrophage behavior and death in embryonic zebrafish during Mycobacterium marinum infection. Steps for the preparation of bacteria, infection of the embryos, and intravital microscopy are included. This technique may be applied to the observation of cellular behavior and death in similar scenarios involving infection or sterile inflammation.

Zebrafish is an excellent model organism for studying innate immune cell behavior due to its transparent nature and reliance solely on its innate immune ...

Brain Ventricular Microinjections of Lipopolysaccharide into Larval Zebrafish to Assess Neuroinflammation and Neurotoxicity

Neuroinflammation is a key player in various neurological disorders, including neurodegenerative diseases. Therefore, it is of great interest to research and develop alternative in vivo neuroinflammation models to understand the role of neuroinflammation in neurodegeneration. In this study, a larval zebrafish model of neuroinflammation mediated by ventricular microinjection of lipopolysaccharide (LPS) to induce an immune response and neurotoxicity was developed and validated. The transgenic zebrafish lines elavl3:mCherry, ETvmat2:GFP, and mpo:EGFP were used for real-time quantification of brain neuron viability by fluorescence live imaging integrated with fluorescence intensity analysis. The locomotor behavior of zebrafish larvae was recorded automatically using a video-tracking recorder. The content of nitric oxide (NO), and the mRNA expression levels of inflammatory cytokines including interleukin-6 (IL-6), interleukin-1&#946; (IL-1&#946;), and human tumor necrosis factor &#945; (TNF-&#945;) were investigated to assess the LPS-induced immune response in the larval zebrafish head. At 24 h after the brain ventricular injection of LPS, loss of neurons and locomotion deficiency were observed in zebrafish larvae. In addition, LPS-induced neuroinflammation increased NO release and the mRNA expression of IL-6, IL-1&#946;, and TNF-&#945; in the head of 6 days post fertilization (dpf) zebrafish larvae, and resulted in the recruitment of neutrophils in the zebrafish brain. In this study, injection of zebrafish with LPS at a concentration of 2.5-5 mg/mL at 5 dpf was determined as the optimum condition for this pharmacological neuroinflammation assay. This protocol presents a new, quick, and efficient methodology for brain ventricle microinjection of LPS to induce LPS-mediated neuroinflammation and neurotoxicity in a zebrafish larva, which is useful for studying neuroinflammation and could also be used as a high-throughput in vivo drug screening assay.

This protocol demonstrates the microinjection of lipopolysaccharide into the brain ventricular region in a zebrafish larval model to study the resulting neuroinflammatory response and neurotoxicity.

Neuroinflammation is a key player in various neurological disorders, including neurodegenerative diseases. Therefore, it is of great interest to research ...

Neuroscience

Monitoring On-Target Signaling Responses in Larval Zebrafish - Z-REX Unmasks Precise Mechanisms of Electrophilic Drugs and Metabolites

Reactive metabolites and related electrophilic drugs are among the most challenging small molecules to study. Conventional approaches to deconstruct the mode of action (MOA) of such molecules leverage bulk treatment of experimental specimens with an excess of a specific reactive species. In this approach, the high reactivity of electrophiles renders non-discriminate labeling of the proteome in a time- and context-dependent manner; redox-sensitive proteins and processes can also be indirectly and often irreversibly affected. Against such a backdrop of innumerable potential targets and indirect secondary effects, linking phenotype to specific target engagement remains a complex task. Zebrafish targeting reactive electrophiles and oxidants (Z-REX)-an on-demand reactive-electrophile delivery platform adapted for use in larval zebrafish-is designed to deliver electrophiles to a specific protein of interest (POI) in otherwise unperturbed live fish embryos. Key features of this technique include a low level of invasiveness, along with dosage-, chemotype-, and spatiotemporally-controlled precision electrophile delivery. Thus, in conjunction with a unique suite of controls, this technique sidesteps off-target effects and systemic toxicity, otherwise observed following uncontrolled bulk exposure of animals to reactive electrophiles and pleiotropic electrophilic drugs. Leveraging Z-REX, researchers can establish a foothold in the understanding of how individual stress responses and signaling outputs are altered as a result of specific reactive ligand engagement with a specific POI, under near-physiologic conditions in intact living animals.

Zebrafish targeting reactive electrophiles and oxidants (Z-REX) is a chemical biology-based method for the investigation of reactive small-molecule signaling. This technique can be applied to live fish of different developmental stages. Here, we couple standard assays in zebrafish with Z-REX for signaling pathway analysis.

Reactive metabolites and related electrophilic drugs are among the most challenging small molecules to study. Conventional approaches to deconstruct the ...

Biology

Targeted Liver Ablation: Inducing Hepatocyte Death with Metronidazole in Transgenic Zebrafish Larvae

Source: Choi T.Y., et al. <a href="https://www.jove.com/v/52785/" target="_blank">Hepatocyte-specific Ablation in Zebrafish to Study Biliary-driven Liver Regeneration</a>. J. Vis. Exp. (2015)
The article describes targeted liver ablation, a method for inducing hepatocyte death with metronidazole in transgenic zebrafish larvae. The sample protocol describes the process for generating, manipulating and assessing targeted liver ablation in zebrafish.

Source: Choi T.Y., et al. Hepatocyte-specific Ablation in Zebrafish to Study Biliary-driven Liver Regeneration. J. Vis. Exp. (2015)
The article describes ...

JoVE Encyclopedia of Experiments

Danio rerio (zebrafish)

Larva

A zWEDGI Technique to Visualize Fungal Pathogen-Infected Zebrafish Larvae

Source: Thrikawala, S. et al., <a href="https://app.jove.com/v/61165">Infection of Zebrafish Larvae with Aspergillus Spores for Analysis of Host-Pathogen Interactions.</a>&#160;J. Vis. Exp.&#160;(2020)
The video demonstrates a zebrafish wounding and entrapment device technique for observing fluorescently labeled fungal pathogen infection in zebrafish larvae. Initial infection attracts macrophages, and as infection persists, neutrophil recruitment progresses at the injection site.

1. Preparation of&#160;Aspergillus&#160;spores for injection

	From an&#160;Aspergillus&#160;spore suspension, calculate the volume needed to obtain 1 x ...

Immunology

Immunodiagnostics

Imaging and Visualization Techniques

A Technique to Quantify Fungal Infection in Zebrafish Larvae

Source: Thrikawala, S., et al. <a href="https://app.jove.com/v/61165">Infection of Zebrafish Larvae with Aspergillus Spores for Analysis of Host-Pathogen Interactions.</a> J. Vis. Exp. (2020).
This video demonstrates a method to quantify fungal infection in zebrafish larvae. Fungal spores are microinjected into the larval hindbrain ventricle, and the larvae are incubated to allow infection development. Infected larvae are harvested at defined intervals, and infection progress is assessed by enumerating colony-forming units.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Targeting Drugs to Larval Zebrafish Macrophages by Injecting Drug-Loaded Liposomes

Samenvatting

Meer video's verkennen

Hoofdstukken in deze video