Materials and Reagents

Glass vials, 5 ml (VWR international, catalog number: 548-0555)
Polycarbonate membranes, 400 and 100 nm pore size (Nucleopore Track-Etch membranes, Whatman, catalog numbers: 7065257, 6257028)
Borosilicate glass microneedles with filament, 10 cm (Science Products, Sutter Instruments, catalog number: BF100-78-10)
Microloader 20 μl (Eppendorf, catalog number: 5242956003)
Disposable Petri dishes, 92 × 16 mm with cams (SAERSTEDT, catalog number: 82.1473.001)
Plastic transfer pipettes (SAERSTEDT, catalog number: 86.1171)
Glass bottom dishes (WillCo-dish, catalog number: GWST-5040)
Zebrafish breeding tanks with divider (Tecniplast, Italy)
Adult zebrafish (wildtype (AB/TL) or transgenic line of interest)
Low-melting-point agarose (Sigma-Aldrich, catalog number: A9414)
Instant Ocean sea salt for aquariums (Instant Ocean, catalog number: SS15-10)
Fluorescent nanoparticles (e.g., liposomes as described below; store in the dark)
Lipids (Avanti Polar Lipids, Lipoid GmbH, and/or Sigma-Aldrich)
Chloroform (Sigma-Aldrich, catalog number: 67-66-3)
(Optional) Ethanol (Honeywell, catalog number: 67-63-0)
(Optional) N-Phenylthiourea (PTU) (Sigma-Aldrich, catalog number: P7629; see Recipes; store at room temperature)
Tricaine (ethyl 3-aminobenzoate methanesulfonate) (Sigma-Aldrich, catalog number: A5040) (see Recipes; store at 4°C and in the dark once diluted)
Formulation buffer (e.g., HEPES 10 mM; see Recipes; store at room temperature)
Egg water (see Recipes)
Agarose gel (see Recipes)

Equipment

Stainless steel tip tweezers (IDEAL-TEK, catalog number: 3480641)
Mini-extruder (Avanti Polar Lipids, catalog number: 610000)
Syringes 1000 µl (Avanti Polar Lipids, catalog number: 610017)
Vacuum desiccator (Fisher Scientific, model: Pirex 1594/02D)
Bench-top vortex (Scientific Industries, model: G-560E)
Nanosizer (Malvern Zetasizer Nano ZS)
LED-UV light source (wavelength 370 nm, FWHM = 13.4 nm; H2A1-H375-S, Roithner Lasertechnik)
Stereo microscope (Leica, model: MS5)
Micropipette puller (Sutter Instruments, model: P-97)
Injector (Eppendorf, FemtoJet, catalog number: 524702135) attached to a manual micromanipulator (World Precision Instruments, WPI model M3331R) on a steel base plate (WPI, code 5052) with a magnetic stand (WPI, code M10)
Incubator (Heraeus, model: B15)
Water bath (ELBANTON, Julabo, model: MWB)
Fluorescent stereo microscope (Leica, model: M205 FA-2)
Confocal microscope (Leica Microsystems, model: SP8/SPE)
Fully approved zebrafish facility (see Ethics section below)

Software

Zetasizer Software, version 7.13
Fiji distribution of ImageJ, versions 1.51p and 1.52p (Schindelin et al., 2012; Schneider et al., 2012)
Confocal microscopy data were processed using Leica Software (Leica Application Suite X software, version 3.5.5.19976)

Procedure

Figure 1. Protocol for intravenous nanoparticle administration, imaging, and analysis in zebrafish embryos. A. Nanoparticle formulation, e.g., extrusion of hydrated lipid solution. Following formulation and biophysical characterization, liposomes should be stored at 4°C. *CryoEM is optional but recommended. Immediately prior to injection, confirm, at the very least, nanoparticle size. Wherever possible, we recommend using freshly prepared nanoparticles. If this is not possible, confirm nanoparticle stability over time. B. Zebrafish microinjection. Cross adult zebrafish (male and female, specific transgenic line if required) to obtain zebrafish eggs by external fertilization. Raise embryos at 28.5°C until the desired stage (i.e., ~56 h post fertilization, hpf). Remove the chorion if embryos have not yet hatched. Mount embryos with agarose gel (0.4% agarose + 0.01% tricaine). Intravenously inject (fluorescently labeled) nanoparticles within the sinus venosus/duct of Cuvier (at 2 dpf). For more details about the injection site, see Figure 2. C. Confocal imaging nanoparticle biodistribution. Use 10× objective to visualize whole body nanoparticle distribution (3-4 overlapping images), 40× objective to visualize tissue level distribution (example shows tail region containing scavenger endothelial cells of the CHT and CV as well as blood resident macrophages), and 63× objective to visualize cellular level distribution (example shows liposomes accumulated within macrophages). Liposomes in cyan, macrophages in magenta, and fluorescently labeled hyaluronic acid (as a marker for scavenger endothelial cells) in yellow. CV, caudal vein; CHT, caudal hematopoietic tissue; DA, dorsal aorta; DLAV, dorsal longitudinal anastomotic vessel; and ISV, intersegmental vessels. Scale bars: 200 µm (10×), 50 µm (40×), and 25 µm (10×). Lateral view images are adapted from Arias-Alpizar et al. (2020) .

Formulation of fluorescently labeled nanoparticles
1. Prepare liposomes (or other nanoparticles) using a preferred method [for example, extrusion, ethanol injection, or microfluidic preparation (Yu et al., 2009; Zhang, 2017) for lipid nanoparticles]. For fluorescently labeled nanoparticles, ensure that the incorporated dye does not adversely affect nanoparticle biophysical properties but can be easily detected above background autofluorescence within the embryo.
  Preparation of photoactive liposomes [containing 1 mol% fluorescent lipid probe, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl), DOPE-LR] by extrusion is briefly described. Any aqueous buffer can be used.
2. Formulate liposomes in 10 mM HEPES buffer at a total lipid concentration of 1-5 mM. Generally, liposomes can be formulated by manual extrusion up to a lipid concentration of approximately 30 mM. Final liposome concentration is typically reported as a total lipid concentration.
  1. Prepare individual lipid stock solutions (1-10 mM) in chloroform.
  2. In a glass vial, combine lipids to the desired molar concentration and dry to a film, first under a stream of N₂ then >1 h under vacuum using a bench-top vacuum desiccator. This is to ensure complete removal of residual chloroform. In the highlighted study, various molar ratios of lipids were used. For instance, to test the effect of increasing the positive surface charge [Figure 2 of Arias-Alpizar et al. (2020)], liposomes were formulated at 1:1, 1:3, and 1:9 molar ratios of cationic lipids (1-3) and zwitterionic 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC).
  3. Manually extrude above phase transition temperature (Tm) of all lipids to form large unilamellar vesicles.
    1. Hydrate lipids to create a suspension. Add aqueous buffer to the lipid film to achieve the desired final lipid concentration.
      Note: In the setup described, we recommend using 0.5-1 ml to avoid sample loss in the ‘dead’ volume (approximately 50 µl) within the mini-extruder block.
    2. Warm solution above Tm of all lipids. Vortex the solution vigorously to create a homogenous lipid suspension. There should be no visible film or visible aggregates remaining in the solution. Depending on the lipid mixture and concentration, a lipid suspension may appear cloudy or transparent.
    3. Load suspension into syringe and fit to one side of extruder block. Fit a clean, empty syringe on the other side of extruder block. Heat extruder block to desired temperature (i.e., 5-10°C above Tm of all lipids).
    4. Pass 11 times through 2 × 400 nm polycarbonate (PC) membranes; always end in a clean syringe to avoid particulate contamination (e.g., dust).
    5. Pass 11 times through 2 × 100 nm PC membranes.
    6. Collect the formulated liposomes in a clean glass vial and store in the dark at 4°C until further use; time depends on liposome stability. We highly recommend using freshly prepared liposomes; however, if using stored liposomes, always confirm size (DLS measurement) immediately prior to use.
  4. (Optional) To analyze light-activated liposomes in vitro, irradiate liposomes (370 ± 7 nm, 202 mW cm^-2) in quartz cuvettes with the LED mounted at a distance of 1 cm from the sample.
3. Characterize the physicochemical properties of the (freshly prepared) liposomes. Size and surface charge are measured using a Malvern Zetasizer Nano ZS. If using stored liposomes, always confirm size immediately prior to injection in embryonic zebrafish.
4. (Optional) CryoEM analysis to confirm liposome morphology, size, and structure (Almgren, 2000; Frederik and Hubert, 2005; Crawford et al., 2011; Baxa, 2018) is strongly advised for any new nanoparticle, in particular, if unusual/unexpected biodistribution is observed in the zebrafish embryo.

Preparation of zebrafish embryos
Note: These steps require a specific license and corresponding approval by regulatory authorities (see Ethics section below).
1. Breed adult zebrafish by setting up crosses in pairs in breeding tanks during the afternoon, separating male and female with a plastic and transparent divider, and use a lid to prevent the zebrafish from escaping from the tank.
2. The following morning, remove the divider at the beginning of the light period to allow and control breeding time.
3. Collect zebrafish eggs with a strainer and transfer to Petri dishes containing egg water (see Recipes) (~50-70 embryos per Petri dish).
4. Remove unfertilized eggs (remain at a single stage and acquire an irregular shape over time) or dead embryos.
5. Raise eggs in an incubator at 28.5°C. Replenish with fresh egg water every day.
6. (Optional) Add 25 μl PTU to a Petri dish containing 50 ml egg water and around 50-70 embryos to prevent pigmentation at ~24 h post fertilization.

Microinjection of zebrafish embryos/larvae
1. At 56 h post fertilization (hpf), remove the chorion protecting zebrafish embryos if they have not hatched yet. Under a microscope, use a pair of stainless steel tip tweezers to carefully remove the chorion by making a tear in it and pulling opposite sides until it is removed and the embryo is released. Avoid direct contact of tweezers with the embryo to avoid damaging it.
  Note: Pronase treatment is a common alternative for dechorionation; however, we recommend the use of tweezers to avoid unnecessary exposure of the embryo to proteolytic enzymes. If high throughput is required, automated chorion removal using pronase can be performed (Mandrell et al., 2012).
2. Prepare an agarose solution (0.4% agarose in egg water and add tricaine, 0.01% final concentration) to embed the zebrafish embryo. Submerge container in a water bath at 36-40°C to prevent agarose gelation.
  Caution: Be careful with the temperature control at this step because overheating can damage the zebrafish embryo during the mounting steps.
3. Prepare injection microneedles by pulling glass needles with filament according to the instructions of the micropipette puller machine.
4. Transfer approximately 20 embryos, anesthetized in 0.01% tricaine, with a plastic transfer pipette and place them in a clean, empty plastic Petri dish.
5. Remove any excess egg water and pipette in prepared agarose solution (~3 ml). Embryos should be evenly distributed and preferably on the bottom surface of the Petri dish. Carefully use tweezers to manipulate embryos into the desired position (preferably in lateral view; see Figure 2 and suggested Videos 1 and 2 for more detail). Allow agarose gel to cool for a few minutes to solidify.
  
  Figure 2. Microinjection site of zebrafish embryo/larvae. A. Schematic of a zebrafish embryo showing intravenous injection site within the sinus venosus/duct of Cuvier at 56 hpf. B. Zebrafish embryo Tg(kdrl:GFP) after injection of fluorescently labeled liposomes (in magenta/gray) at 56 hpf, 1 hpi. Volume of injection: 1 nl. Scale bar 200 µm (10×).
  
  Video 1. Successful injection zebrafish embryo 2 dpf.
  
  Video 2. Unsuccessful injection zebrafish embryo 2 dpf.
6. Proceed with the microinjection.
  1. Load fluorescently labeled nanoparticles (~3 μl) in a glass microneedle with a pipette and insert needle into the arm of the injector.
  2. Calculate the injection volume (1 nl volume is recommended).
    1. Cut the length of the microneedle with stainless steel tip tweezers (removing a section of the thinner part of the pre-pulled microneedle tip).
    2. Press the foot pedal to expel one droplet.
    3. Measure the size of a droplet under the microscope. The incorporation of a scale into the ocular of the microscope (or a stage micrometer, e.g., agar scientific AGL4079, as an alternative) allows the measurement of a droplet equivalent to 1 nl (i.e., 100 μm at 40×).
    4. Adapt the volume accordingly by adjusting the micromanipulator setting (i.e., pressure).
      Note: Repeat this step periodically during injections to ensure injection volume is consistent.
  3. Position the microneedle towards the sinus venosus/duct of Cuvier (Figure 2).
    Note: To reach the zebrafish embryo, the needle must be carefully moved ‘in and out’ once inserted in the agar.
  4. Penetrate the skin with the injection needle.
    Note: Be careful not to touch the yolk sac with the tip needle to avoid over-insertion of the microneedle that can lead to unsuccessful i.v. injection.
  5. Once the needle is inside the duct of Cuvier, gently pull the needle back, creating a small pyramidal space, and inject nanoparticle solution (1 nl). At the time of injection, a backward flow/spreading of circulating blood cells should be observed (see Videos 1 and 2 above to differentiate between successful and unsuccessful injection).
  6. Once injected, carefully remove the microneedle from the embryo.
7. Release the zebrafish embryo from the agarose gel. First, add egg water (~5 ml) on top of the agarose gel. Second, use tweezers to carefully separate the embryo from the agarose (without touching the embryo).
8. Once the embryo is released and able to swim, transfer the injected fish with a plastic transfer pipette to a clean Petri dish containing fresh egg water and 0.01% tricaine to proceed with screening for successfully injected fish.

Screening of successfully injected embryos
1. Make a selection of well-injected zebrafish embryos under a fluorescent stereo microscope. Well-injected embryos show fluorescent nanoparticles in circulation (Figure 3A). Discard any embryo that does not show nanoparticles in circulation, i.e., injected in the yolk sac, pericardial space, etc. (Figures 3B-3D).
2. Ensure fish look healthy with no physical damage (i.e., from injection needle).
  
  Figure 3. Selection of injected zebrafish embryos. A. Successfully injected zebrafish embryos; nanoparticles are clearly in circulation, homogeneously distributed throughout the vasculature of the embryo. B-D. Unsuccessful injections; nanoparticles accumulate in the pericardial space (B, zebrafish embryo in ventral view) or in the yolk sac (C, within dashed circles) or a combination of both (D), and only partial volume of nanoparticles are in circulation. Do not use these embryos as representative of nanoparticle biodistribution. Note: For clarity, confocal images are shown here. Screening for successful injection should be done quickly under a stereo microscope. E. Nanoparticles in circulation (in magenta, within boxes) in the dorsal aorta. Intensity values measured in this area can be used to calculate the liposome circulation life at different time points (i.e., 1-24 h post-injection, hpi). We recommend using n = 6 for each point. All zebrafish embryos shown are 2 days post fertilization (dpf); Tg (kdrl:GFP) 1 hpi. Scale bars: 200 µm (A-D), 50 µm (F).

Transfer the zebrafish embryo with a plastic transfer pipette to a Petri dish containing fresh egg water (with no tricaine added) and keep the zebrafish embryo at 28.5°C until further use (i.e., imaging).

(Optional step) Applying UV light after intravenous injection of photoactive liposomes
In this case, a LED (375-nm) lamp driven by a custom-built LED driver (I = 350 mA) is used as UV light source.
1. Determine the power density (in mW cm^-2) of the light source and calculate light doses (J per embryo) by multiplying the optical power density by the irradiation time.
2. For embryo irradiation, the UV source (wavelength 370 ± 7 nm) was positioned approximately 1.5 cm above the agar-embedded embryo (~90 mW cm^-2) (Arias- Alpizar et al., 2020). A minimum irradiation dose of ~2.4 J per embryo was used.

Embedding of embryos/larvae in agarose solution (0.4% + 0.01% tricaine) for confocal imaging
1. Use a glass bottom dish suitable for confocal imaging.
  Note: This must be able to contain liquid agarose before gel formation, and therefore generic glass slides are not suitable.
2. Randomly select at least six successfully injected zebrafish embryos to image and transfer them with a plastic transfer pipette into a glass bottom plate.
3. Place ~3 ml of agarose solution into the glass bottom dish. Make sure the agarose covers the surface of the whole bottom dish and gently position the zebrafish embryos into the correct position for desired imaging (i.e., lateral, dorsal) with the help of the tweezers. Allow the gel to cool and solidify over a few minutes.
4. Mounted embryos (i.e., in dorsal or lateral view) can now be used for imaging. We recommend adding egg water on top of the agarose to prevent the sample from drying out if the time of imaging is extensive (>3 h). After imaging, transfer the fish into Petri dishes containing fresh egg water and allow to swim freely at 28.5°C for at least ~24 h to monitor possible effects after injection and/or for further analysis.

Confocal imaging of nanoparticle biodistribution and identification of specific cellular interactions
For nanoparticle assessment, we recommend using transgenic zebrafish lines Tg (kdrl:GFP) (Jin et al., 2005) and Tg (mpeg1:GFP) (Ellett et al., 2011) to identify endothelial cells and macrophages, respectively.
Note: The fluorescent nanoparticle probe must not overlap with transgenic fluorescence emission.
1. Capture confocal z-stacks using a 10× air objective (HCX PL FLUOTAR), a 40× water-immersion objective (HCX APO L), or 63× water-immersion objective (HC PL APO CS).
  1. For whole-embryo views, maximum projections of three overlapping z-stacks (10×) were captured to cover the complete embryo.
  2. Figures 3, 4, 6, and 7c and Supplementary Figures 1-3, 7-9 of Arias-Alpizar et al. (2020) illustrate the use of confocal imaging after intravenous administration of liposomes in zebrafish embryos.

Identification of nanoparticle interactions with scavenger endothelial cells (SECs, i.e., analogous to liver sinusoidal endothelial cells in mammals) can be assessed using fluorescent hyaluronic acid (fluoHA) as a specific and non-competitive marker of SECs (Figure 1C) (Campbell et al., 2018). In addition, the involvement of the key scavenger receptors, Stabilin-2 and -1, can be easily assessed through co-injection of dextran sulfate within the embryonic zebrafish (Campbell et al., 2018; Arias-Alpizar et al., 2021). Furthermore, stabilin-2, stabilin-1, and stabilin-1 and -2 knockout embryos have been generated (Campbell et al., 2018; Arias-Alpizar et al., 2021).

Data analysis

Process the images using the Fiji distribution of ImageJ (Schindelin et al., 2012; Schneider et al., 2012). Adjust brightness and contrast, rotate and crop the image if needed. For the whole embryo view, stitch the maximum projection of 3 or 4 images at an appropriate overlapping point (Figure 1C). For comparison and/or quantification, laser intensity, gain, and offset settings must be identical between stacks and between experiments. The quantification of liposome circulation lifetime decay (n = 3-6) shown in Figure 4G and Supplementary Figures 7-8 of Arias-Alpizar et al. (2020) was calculated using a previously described macro for ImageJ (Campbell et al., 2018) using the measured nanoparticle fluorescent intensity within the dorsal aorta, as illustrated in Figure 3E.

Notes

These studies require knowledge, license, and basic skills in zebrafish husbandry and handling. Good zebrafish husbandry practices are critical throughout this protocol to ensure the health and comfort of the zebrafish are maintained at all times. Reproducibility is essential. Repeat all experiments at least twice. Use different zebrafish lines, if possible, and always repeat with new batches of (freshly prepared) nanoparticles. In the case of light application, do not irradiate excessive numbers of embryos simultaneously to avoid embryo-to-embryo variations in light dose. For experiments monitoring changes in liposome biodistribution following light activation, always image the same embryo before and after UV irradiation. Intravenous injections can be performed at any point between 2 and 4 dpf. Ethical approval is required for any experiment in zebrafish after 5 dpf.

Recipes

Egg water
Mix 60 µg of instant Ocean sea salts per ml. Store at room temperature.
N-Phenylthiourea (PTU)
Mix PTU powder in 85% ethanol solution to a final concentration of 3% (w/v). Store at room temperature and in the dark.
Agarose gel
In a clean glass container, mix low melting agar powder in egg water to a final concentration of 0.4%. Melt the agarose mixture in a microwave, shake until dissolved, and keep it in a water bath at 36-40°C. Add tricaine to a final concentration of 0.01%.
HEPES buffer
HEPES (10 mM) adjusted to pH 7.4 with 1 m aqueous NaOH. Ultrapure MilliQ^® water, purified by a MilliQ Advantage A10 water purification system from MilliPore. Store at room temperature.
Tricaine
Mix 400 mg tricaine powder with 97.9 ml of deionized water, and adjust to pH 7 with 2.1 ml of 1 M Tris (pH 9). Store in the dark at 4°C. Mix 1 ml of stock solution with 9 ml of egg water for the working solution.

Acknowledgments

All authors that contributed to the original research paper where this protocol was derived from (Arias-Alpizar et al., 2020) are kindly acknowledged. This work was supported by the Interreg 2 Seas Program 2014-2020 co-funded by the European Regional Development Fund under subsidy contract “Site Drug 2S07-033”.

Competing interests

No competing interests related.

Ethics

All animal experiments must be in accordance with institutional regulations. In this case, zebrafish (Danio rerio, strain AB/TL) were maintained and handled in accordance with guidelines from the European Convention on the protection of vertebrate animals used for experimental and other scientific purposes (Alestrom et al., 2020) and in compliance with the directives of the local animal welfare committee of Leiden University.

References

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