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

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.

1. To prepare liposomes, dissolve 16.4 mg (22.2 µmol) of the phospholipids 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (see Table of Materials), 4.3 mg (11.1 µmol) cholesterol (see Table of Materials) and 2.8 mg (3.7 µmol) 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.
2. 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.
3. Remove the solvent slowly on a rotary evaporator (see Table of Materials) over a temperature controlled water bath set to 45 °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.
4. 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.
5. 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 °C water bath for 20 min in order to form vesicles.
6. 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 °C water bath for 7 min.
7. Extrude liposomes through a membrane using a mini-extruder (see Table of Materials) and 1.0 µm 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 µm^26,27,28.
8. 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 °C and 750 rpm for 30 min using a thermomixer with a 2 mL microcentrifuge tube attachment (see Table of Materials).
9. Ultracentrifuge the mixture at 186,000 x g for 1 h at 4 °C and remove the supernatant in order to remove any unbound polymer or drug. Store the liposomes pellets at 4 °C, protected from light.

2. Characterization of Liposome Size and Zeta Potential

1. 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 °C.
   NOTE: The PDI is a measure of the size distribution of the nanoparticle population. PDI values < 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.

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.

1. Resuspend the liposome pellet in 1 mL of PBS.
2. Remove the supernatant, dilute 10 x with ultrapure water and analyze by high-performance liquid chromatography (HPLC).
3. To determine the drug concentration within the liposomes, add 100 µL of liposome suspension to 900 µL 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.
4. Inject 20 μL of sample into an HPLC system consisting of a quaternary pump, thermostat auto-sampler and a 3 μm 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.
5. 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.

4. Preparation and Injection of Liposomes

1. 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.
2. 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.
3. 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.
4. Set up adult zebrafish breeding pairs the night before and collect embryos as previously described by Rosen et al.²⁹.
5. 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 °C.
6. 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).
7. 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
8. Let the embryos develop at 28.5 °C until 2 days post fertilization (dpf).
9. 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.
10. Anaesthetize the larvae by incubating in E3 media supplemented with 200 µg/mL of Tricaine.
11. 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.
12. 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.
13. Take freshly prepared liposomes (from step 4.3) and back fill an injection needle with approximately 5 μL 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 μg/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 phagosomes^30,31 and lysosomes³², respectively.
14. 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.
15. Cut the tip of the microinjection needle with clean fine tip forceps to generate an opening of approximately 5 μm in diameter.
16. 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).
17. 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.
18. 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.
19. Incubate the larvae at 28.5 °C (protected from light) until analysis by live confocal imaging.

5. Confocal Imaging and Image Analysis to Confirm Macrophage Targeting of Liposomes

1. Heat a water bath to 50 °C.
2. Anaesthetize liposome-injected larvae by transferring to a new Perti dish containing E3 media supplemented with 0.003% PTU and 200 µg/mL Tricaine.
3. 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 °C, add 200 µg/mL of Tricaine.
4. Keep the mounting medium in the 50 °C water bath to prevent premature polymerization of the agarose.
5. 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.
6. 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 µg/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.
7. 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 µg/mL of Tricaine.
8. To live image the hindbrain ventricle on a confocal microscope, use the following settings: 512 x 512 pixels; 40 x 3 μm 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.
9. 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.