This protocol describes a technique for targeting drugs of interest to larval zebrafish macrophages to manipulate and evaluate macrophage function. This technique has advantages over traditional drug delivery strategies such as immersion by ensuring that the drugs are specifically targeted to macrophages through liposome encapsulation. When applied to zebrafish models of disease with an inflammatory component, this technique has the potential to resolve how macrophages contribute to pathological processes.
Here, we demonstrate the utility of this technique by targeting a mitochondrial-reactive oxygen species-inhibiting drug to macrophages to suppress activation. The microinjection of drug-loaded liposomes into larvae can be challenging, as care should be taken to not cause excessive epithelial damage, potentially influencing immunological studies. Visual demonstration of this protocol is important because it involves pharmacological and whole animal live cell methodologies that may not be routinely performed in a single laboratory.
To prepare 10 milligrams of liposomes in a 25-milliliter round-bottom flask, add 31 nanomolars of marine blue and 300 nanomolars of a mitochondrial-reactive oxygen species-inhibiting drug to the flask. Sonicate briefly to fully dissolve, and remove the solvent slowly on a rotary evaporator set over a 45-degree Celsius water bath at 75 rotations per minute under vacuum pressure. When a dry lipid thin film has formed within the glass walls of the flask, further dry the film for 45 minutes under nitrogen to facilitate the complete removal of the organic solvent.
Next, hydrate the film with one milliliter of PBS, and seal the flask with paraffin film before agitating the flask by hand over a 60-degree Celsius water bath for 20 minutes. After seven cycles of freezing and thawing, use a mini-extruder and one-micrometer track-etched polycarbonate membranes to extrude the liposomes for two to six cycles. When large unilamellar vesicles have been obtained, condense the fresh liposomes into a pellet by ultracentrifugation.
Incubate the pellet with one milliliter of 1%poloxamer 188 solution for 30 minutes at 45 degrees Celsius and 750 rotations per minute in a ThermoMixer with a two-milliliter microcentrifuge tube attachment. During the post insertion of poloxamer 188 into the liposomes, the correct poloxamer concentration, incubation time, and temperature are important for achieving the optimal physicochemical characteristics of the resulting liposomes. At the end of the incubation, ultracentrifuge the mixture again under the same centrifuge conditions, and remove the supernatant containing any unbound polymer or drug.
Then store the liposome pellets at four degrees Celsius, protected from light. To determine the size and zeta potential of the liposomes, dilute the liposome formulation with ultrapure water at a one-to-100 ratio. Use a dynamic light scattering analyzer to measure the particle size, polydispersity index, and zeta potential in triplicate at 25 degrees Celsius.
To calculate the entrapment efficiency and drug loading of the liposomes, dissolve the liposomes in Triton X-100. Inject 20 microliters of the dissolved liposome sample into a high-pressure liquid chromatography system with a quaternary pump, a diode array detector set at 230 nanometers, and a three-micrometer, 250-times-4.6-millimeter column. To prepare transgenic zebrafish larvae for the injection, use fine-tip forceps to manually dechorionate 30 hours post-fertilization embryos, and allow the embryos to develop at 28.5 degrees Celsius until two days post-fertilization.
When the embryos have reached the appropriate stage, dilute a freshly prepared liposome formulation at a one-to-one ratio in sterile PBS, and mix the solution for two to three seconds by vortexing. Pour enough 3%methyl cellulose in E3 medium into the lid of a small, 35-millimeter culture dish lid to form a thin film covering the entire surface. Use a plastic transfer pipette to collect a pool of approximately 20 to 25 anesthetized larvae.
Allow the embryos to concentrate at the tip of the pipette by gravity, and carefully transfer the larvae into the middle of the 35-millimeter culture dish lid with minimal solution. Use a microcapillary loader to arrange the larvae with their anteriors toward the top of the injection plate with the dorsal surface facing toward the microinjection needle for injection into the hindbrain ventricle or with their anteriors toward the left of the injection plate with the ventral surfaces facing toward the microinjection needle for injection into the sinus venosus. Use a microcapillary loader to back-fill an injection needle with approximately five microliters of the liposome injection mix.
Mount the needle into a micromanipulator connected to a magnetic stand and a pressure injector, and position the setup under a stereo microscope. Use clean, fine-tip forceps to cut the tip of the microinjection needle to generate an opening of approximately five micrometers in diameter. Set the injector to a pulse duration of approximately 50 milliseconds and an injection pressure of approximately 40 pounds per square inch.
Inject a bolus into a drop of mineral oil positioned over the grid lines of a hemocytometer to calibrate the volume to be injected. Then adjust the pulse duration and/or the injection pressure until the diameter of the bolus covers 2 1/2 of the smallest grid lines. Carefully inject one nanoliter of the liposome injection mix into the hindbrain ventricle of each larvae.
Immediately following the injections, add E3 medium to the injection plate, and use a plastic transfer pipette to gently agitate the larvae out of the methyl cellulose. To image the dorsal surface of the hindbrain on a confocal microscope equipped with a water immersion lens, first fill a 35-millimeter culture dish to a depth of five millimeters with mounting medium, and let the medium polymerize. Excavate a small trench in the agarose of sufficient area to accommodate the yolk sac of the experimental embryo, and use a plastic transfer pipette filled with molten mounting medium to collect an anesthetized larva.
Immediately deposit the larva into the culture dish, and use a microcapillary loader to orientate the yolk sac ventral side down into the excavated holes. Maintain the embryo in this position until the agarose polymerizes. Overlay the embryo with E3 medium supplemented with 200 micrograms per milliliter of tricaine.
To live image the hindbrain ventricle, select the 20x objective, and set the confocal microscope to 512 by 512 pixels and a 2.5x zoom. When the microscope parameters have been set, acquire 40-by-three-micrometer Z-stacks extending from dorsal-most surface of the hindbrain using two-channel imaging to image the blue fluorescent liposomes and green fluorescent macrophages. When all of the images have been obtained, load the files into an appropriate 3D image analysis software program.
Under the Measurement tab, drag the Find Objects protocol into the Drag Tasks Here To Make Measurements space. Select the DAPI channel to highlight the liposomes, and drag the Inspect tool through the Z-sections to detect the liposome-laden macrophages. To quantify the fluorescence intensity of liposomes within the individual cells, drag the Clip To Regions Of Interest protocol into the Drag Tasks Here To Make Measurements space, and use the Rectangle tool to draw a box around the cell of interest in the X, Y, and Z dimensions.
Then select Make Measurement Item from the Measurements dropdown menu to generate a measurement file in the library window. In this table, the size, zeta potential, drug loading, and entrapment efficiency of the produced liposomes are summarized. As observed, the particle size of the liposomes is similar with and without drug loading, although the surface charge of drug-loaded liposomes is slightly more neutral than the control liposomes with marine blue only.
Microinjection of marine blue-labeled liposomes into the hindbrain ventricle, as demonstrated, results in a rapid uptake by resident macrophages that can be readily quantified by confocal microscopy by three hours post-injection. Neutrophils, however, are rarely observed to contain intracellular liposomes. Within individual liposome-laden macrophages, the liposomes accumulate within the phagolysosomal compartments, which is necessary for liposome degradation and subsequent drug content release into the cytoplasm.
Delivery into the sinus venosus can deliver the liposomes to caudal hematopoietic tissue-resident macrophages via the circulation. Microinjection of liposomes loaded with the mitochondrial-reactive oxygen species-inhibiting drug into the hindbrain ventricle can significantly suppress monosodium urate crystal-driven mitochondrial-reactive oxygen species production within liposome-laden hindbrain-resident macrophages. Further validation of the suppressive effects of drugs of interest on macrophage activation states can be performed by investigating proinflammatory cytokine gene expression by whole mount in situ hybridization and the temporal recruitment of neutrophils.
Using this technique, we've been able to target drugs to macrophages to confirm that an immunometabolic mechanism drives their activation in a larval zebrafish model of acute gouty inflammation. Future adaptions of this protocol could include performing surface modification of the liposome to enhance macrophage targeting or to promote targeting to other immune cells, such as neutrophils.
