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 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 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 "toolkit" available to zebrafish researchers wanting to ensure macrophage-targeting of their drugs of interest.
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.
2. Characterization of Liposome Size and Zeta Potential
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.
4. Preparation and Injection of Liposomes
5. Confocal Imaging and Image Analysis to Confirm Macrophage Targeting of Liposomes
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). The surface charge (zeta potential) of drug-loaded liposomes is slightly more neutral when compared with control liposomes, however, they are all negatively charged meaning this will not significantly change their biodistribution pattern.
Microinjection of Marina Blue-labeled liposomes into the hindbrain ventricle results in rapid uptake by resident macrophages that can be readily observed by confocal microscopy by 3 h post injection, when injected into the macrophage lineage-marking transgenic reporter line Tg(mpeg1:EGFP)33 (Figure 2C,E-G). This is in contrast to neutrophils (as marked within the neutrophil-specific Tg(lyz:EGFP)34 reporter line) that are rarely observed containing intracellular liposomes (Figure 2E-G). Within individual liposome-laden macrophages, the liposomes accumulate within phagolysosomal compartments (Figure 2H), which is necessary for liposome degradation and the subsequent release of their drug contents into the cytoplasm35. Selecting different microinjection sites for liposome delivery can impact which tissue-resident macrophages are targeted. As examples, delivery into the hindbrain ventricle efficiently targets hindbrain-resident macrophages (Figure 2D,E) while microinjection into the sinus venosus can deliver the liposomes to CHT-resident macrophages via the circulation (Figure 2I,J).
Consideration of the microinjection site for liposome delivery is important when using this technique to interrogate the macrophage response to an immunological challenge as it helps ensure the particular macrophages under investigation are receiving the drug. We have routinely used this protocol for the delivery of drug-loaded liposomes into the hindbrain ventricle to assess the impact of these drugs on the macrophage response to monosodium urate (MSU) crystals, similarly injected into the hindbrain compartment (Figure 3A). As the causative agent of acute gouty inflammation, MSU crystals activate tissue-resident macrophages to produce pro-inflammatory mediators including Interleukin-1β (IL-1β) through a process dependent upon mitochondrial reactive oxygen species (mROS)10,36,37,38. These activated macrophages then drive neutrophil infiltration, a hallmark of acute gouty inflammation. Microinjection of liposomes loaded with a mROS-inhibiting drug into the hindbrain ventricle can significantly suppress MSU crystal-driven mROS production within liposome-laden hindbrain-resident macrophages (Figure 3B-D). Using the protocol described here, we achieved an entrapment efficiency of 49.12 ± 0.17 %, which resulted in a formulation with a drug concentration of 103.05 ± 0.36 μM. Of note, injecting a 1 nL volume at this concentration resulted in no observable toxicity during our experiments, as evidenced by gross morphological changes or cardiac arrest. Further validation of the suppressive effects of this drug on macrophage activation state can be performed by investigating il1b expression (the zebrafish ortholog of IL-1β) by whole mount in situ hybridization (Figure 4A,B) and the temporal recruitment of neutrophils (Figure 4C,D).
Figure 1: Schematic illustrating conventional free drug delivery versus liposome-mediated drug delivery to larval zebrafish. (A) Strategies routinely used for drug delivery to larval zebrafish are largely limited to immersion in, or microinjection of, free drug. (B) Microinjection of drug-loaded liposomes allows for direct targeting to macrophages, where liposome degradation within phagolysosomal compartments results in cytoplasmic drug delivery. Please click here to view a larger version of this figure.
Figure 2: Targeting drugs to macrophages using fluorescent liposomes. (A) Microinjection set-up showing microinjection needle (black arrow) and larvae arrayed in a 35 mm tissue culture dish within 3% methylcellulose. (B) Magnified view of larvae arrayed as in A. (C) Live confocal image of 2 dpf Tg(mpeg1:EGFP) larvae, anterior to left. (D) Magnified view of arrayed larvae, as in A, demonstrating microinjection into the hindbrain ventricle (black arrow marks microinjection needle). (E) Schematic illustrating targeting of hindbrain-resident macrophages and live confocal images (dorsal views, anterior to left) of the hindbrain region of Tg(mpeg1:EGFP) and Tg(lyz:EGFP) larvae, 3 h following hindbrain microinjection of Marina Blue-labeled liposomes (white arrows mark liposome-laden macrophages). (F and G) Quantification of liposome uptake by macrophages and neutrophils (as detected in E), measured as the number of cells containing (F) and the fluorescence intensity/cell (G) of Marina Blue-labeled liposomes. (H) Live confocal image of liposome-laden macrophage within the hindbrain marked with a red fluorescent marker of phagosomes and a far red fluorescent marker of lysosomes within Tg(mpeg1:EGFP) larvae, 3 h following hindbrain microinjection of Marina Blue-labeled liposomes. (I) Magnified view of arrayed larvae, as in A, demonstrating microinjection into the sinus venosus (black arrow marks microinjection needle). (J) Schematic illustrating targeting of CHT-resident macrophages and live confocal images (lateral views, anterior to left) of the CHT region of Tg(mpeg1:EGFP) and Tg(lyz:EGFP) larvae, 3 h following microinjection of Marina Blue-labeled liposomes into the sinus venosus (white arrows mark liposome-laden macrophages). Error bars display mean ± SD. *p<0.05; ****p < 0.0001, Student's t-test. Scale bars = 100 mm (A), 100 μm (B), 250 μm (C, D, and I), 10 μm (E), 5 μm (H), 50 μm (J). This figure has been modified from previous publication24. Please click here to view a larger version of this figure.
Figure 3: Microinjection of liposomes loaded with a mROS-inhibiting drug suppresses mROS production within activated macrophages. (A) Schematic illustrating the targeting of a mROS-inhibiting drug to macrophages and assessing it's impact on macrophage activation following MSU crystal stimulation. (B and C) Live confocal images of liposome-laden macrophages (control liposomes/L-C (B) or mROS-inhibiting liposomes/L-MI (C)) within the hindbrain of Tg(mpeg1:EGFP) larvae, also marked with a fluorescent mROS-specific probe (see Table of Materials, where the fluorescent signal is displayed as a heatmap with warm colors representing higher levels of mROS), 3 h following hindbrain microinjection of Marina Blue-labeled liposomes and MSU crystals. Marina Blue fluorescence is pseudo-colored in grayscale. (D) Quantification of fluorescence intensity of mROS-specific probe within macrophages, as detected in B and C. Error bars display mean ± SD. ****p < 0.0001, Student's t-test. Scale bar = 10 μm (B). This figure has been modified from previous publication24. Please click here to view a larger version of this figure.
Figure 4: Microinjection of liposomes loaded with a mROS inhibitor suppresses il1b expression within activated macrophages and macrophage-driven neutrophil recruitment. (A) Expression of il1b (marked by black arrows), as detected by whole mount in situ hybridization, within the hindbrain region 3 h following hindbrain microinjection of control (L-C) or mROS-inhibiting liposomes (L-MI) and MSU crystals, anterior to left. Numbers represent proportion of larvae with displayed phenotypes. (B) Quantification of il1b expression, as detected in A, shown as percent larvae demonstrating high, low or no expression. (C) Confocal images of neutrophils within the hindbrain region of Tg(lyz:EGFP) larvae (dorsal views, anterior to left), as detected by immunofluorescence 3 (3 hpi) and 6 (6 hpi) h following hindbrain microinjection of L-C or L-MI and MSU crystals. (D) Temporal quantification of neutrophils, as detected in C, 1 (1 hpi), 3 (3 hpi), 6 (6 hpi) and 9 (9 hpi) h following hindbrain microinjection of L-C or L-MI and MSU crystals. Error bars display mean ± SD. ****p < 0.0001, n.s. not significant, one-way ANOVA, Dunnett's post hoc test. Scale bars = 100 μm (A), 50 μm (C). This figure has been modified from previous publication24. Please click here to view a larger version of this figure.
Physicochemical property of liposomes (L) | Control liposomes | mROS-inhibiting liposomes |
Size (μm) | 1.2 ± 0.07 | 1.1 ± 0.04 |
Zeta potential (mV) | -25.9 ± 0.57 | -13.0 ± 0.11 |
Entrapment efficiency (EE, %) | N/A | 49.12 ± 0.17 |
Drug loading (DL, %) | N/A | 0.37 ± <0.01 |
Table 1: Physicochemical characteristics of PBS (control) or mROS-inhibiting liposomes (data are means ± standard deviation, n = 3). All liposomes were labeled with Marina Blue.
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 used to target innate immune cells in larval zebrafish. These include targeting the antimalarial drug rifampicin to Mycobacterium marinum-infected macrophages to promote bacterial clearance using sub-micron size polymeric nanoparticles39. In another example, encapsulation of (R)-roscovitine, an inducer of neutrophil apoptosis, within polymerosomes has been shown to target this drug to neutrophils, thereby promoting inflammation resolution40. In this protocol, we have used liposomes as a vehicle for drug delivery due to their non-toxic, biocompatible and biodegradable properties. In addition, they are non-immunogenic, which is of particular importance when used to investigate immunological responses, such as those described here. A range of cargos can also be carried including hydrophilic, hydrophobic, amphipathic and lipophilic drugs.
When performing this protocol, there are a number of critical steps where special care must be taken. These include steps where larvae are physically handled and care must be taken to minimize any unintentional damage to the larvae. This includes when manually dechorionating the larvae (especially avoiding contact with the delicate epithelial lining of the yolk), arraying larvae onto the injection plate, their removal from the plate following injections and their mounting for live confocal imaging. An unavoidable component of this protocol is the need to delivery the liposomes (and any immune challenges) through microinjection which can cause local inflammation and therefore may influence the experimental outcome. Microinjection into larval zebrafish does require a certain degree of training to minimize tissue damage. In our work, generating a microinjection needle tip diameter of approximately 5 μm generates very minor surface epithelial damage (as evidenced by very low expression of the inflammatory marker matrix metallopeptidase 9 within surface epithelial cells in the immediate vicinity of the microinjection wound10) when injecting into the hindbrain ventricle of 2 dpf larvae. This diameter tip is also small enough to avoid leakage of injected contents, out of the hindbrain ventricle, when the microinjection needle is removed. It is important to note here that injection volumes greater than 2 nL will often result in some of the injected contents overflowing through the injection hole as a result of exceeding the volume of the ventricle. A tip diameter of 5 μm is also of sufficient size to inject subsequent immune challenges, such as MSU crystals10. Beveling the microinjection needle tip to 45° with a microgrinder so as to generate a sharp tip may provide better penetration of the outer periderm and inner basal epithelial layers covering the hindbrain ventricle41. It is important to note that control injections like those used here (i.e., control liposomes) are essential when assessing the effects of drug-loaded liposomes on macrophage function to control for the injection process itself. Selecting a concentration for a given drug when formulating into liposomes is also an important step as established efficacious drug concentrations, when delivered by immersion, might differ from those necessary when using liposome-mediated drug delivery. In addition, when using this protocol, one should be careful not to alter the physicochemical characteristics of the liposomes post modification with poloxamer as this may affect their biodistribution pattern.
An area for modification and future development of this protocol will be to investigate incorporating different surface features to the liposomes such as ligands or receptors to further enhance macrophage targeting and uptake, or to target different immune cells, such as neutrophils. This advanced approach will require further studies to uncover surface features that are unique to the different zebrafish immune cell compartments, which are currently poorly understood. Further areas for modification of this protocol include the incorporation of other fluorescent probes into the liposomes to expand their versatility (e.g., when injected into other transgenic reporter lines) and altering their physical properties such size and charge42,43. It will also be important to examine whether different routes of liposome administration at different larval stages can expand their versatility to target additional macrophage populations, for example intestinal epithelial cells. In the protocol detailed here, all injections were performed on 2 dpf larvae. During the third day of zebrafish development a single continuous lumen from the mouth to anus forms, following the opening of the mouth44. By the forth day the anus opens resulting in a completely open-ended tube lined with a polarized epithelium44. It will be interesting to see if immersion of older larvae in liposomes can facilitate the targeting of encapsulated drugs to intestinal epithelial cells.
The authors have nothing to disclose.
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.
1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) | Avanti Polar Lipids, Inc. | 850355P | |
1,2-diseteroyl-sn-glycero-3-phosphocholine (DSPE) | Avanti Polar Lipids, Inc. | 850367P | |
1.0 µm Whatman Nuclepore Track-Etched polycarbonate membranes | GE Healthcare Life Sciences | 110610 | |
25 mL round-bottom flask | Sigma-Aldrich | Z278262 | |
35 mm culture dish | Thermo Scientific | 150460 | |
Acetonitrile | Sigma-Aldrich | 34998 | |
Agilent 1260 Infinity Diode Array Detector | Agilent Technologies | G4212B | |
Agilent 1260 Infinity Quaternary Pump | Agilent Technologies | G1311B | |
Agilent 1290 Infinity Series Thermostat | Agilent Technologies | G1330B | |
Avanti mini-extruder Avanti Polar Lipids Inc. | Avanti Polar Lipids Inc. | ||
borosilicate microinjection needles | Warner Instruments | 203-776-0664 | |
CaCl2 | Sigma-Aldrich | C4901-100G | |
cholesterol | Sigma-Aldrich | C8667 | |
Dumont No.5 fine tip forceps | Fine Science Tools | 11251-10 | |
Eppendorf Microloader pipette tip | Eppendorf | 5242956003 | |
Eppendorf SmartBlock 1.5 mL, thermoblock for 24 reaction vessels | Eppendorf | 4053-6038 | |
eyelash manipulator | Ted Pella Inc. | 113 | |
hemocytometer | Hawksley | BS.748 | |
HEPES | BDH Chemicals | 441474J | |
HPLC system | Agilent Technologies | 1260 series HPLC system | |
KCl | Sigma-Aldrich | P9541-1KG | |
low melting point agarose | Invitrogen | 16520-100 | |
LysoTracker Deep Red | Invitrogen | L12492 | 1 mM stock solution in DMSO, keep at -20 °C and protect from light. |
LysoTracker Deep Red | Thermo Scientific | L12492 | |
magnetic stand | Narishige | GJ-1 | |
Marina Blue 1,2-dihexadecanoyl-sn-glycero-phosphoethanolamine (Marina Blue DHPE) | Invitrogen | M12652 | Keep at -20 °C and protect from light. |
Methanol | Sigma-Aldrich | 34860 | |
methyl cellulose | Sigma-Aldrich | M0387-500G | |
methylene blue | Alfa Aesar | 42771 | |
MgSO4 | Sigma-Aldrich | 230391-500G | |
micromanipulator | Narishige | M-152 | |
mineral oil | Sigma-Aldrich | M-3516 | |
Mitochondria-targeting antioxidant MitoTEMPO | Sigma-Aldrich | SML0737 | |
MitoSOX Red Mitochondrial Superoxide Indicator | Thermo Scientific | M36008 | |
MitoTEMPO | Sigma-Aldrich | SML0737 | Keep at -20 °C and protect from light. |
N-Phenylthiourea (PTU) | Sigma-Aldrich | P7629-10G | Take care when handling, toxic. |
NaCl | BDH Chemicals | 27810.295 | |
PBS (pH 7.4) | Gibco | 10010-023 | |
Petri dish (100 mm x 20 mm) | Corning Inc. | 430167 | |
Phenomenex C18 Gemini-NZ 3 mm 250 mm x 4.6 mm column | Phenomenex | 00G-4439-E0 | |
pHrodo Red Escherichia coli BioParticles Conjugate | Thermo Scientific | P35361 | |
pHrodo Red Escherichia coli BioParticles Conjugate | Invitrogen | P35361 | Keep at -20 °C and protect from light. Make 1 mg/mL stock solution by dissolving 2 mg lyophilized product in 2 mL of PBS supplemented with 20 mM HEPES, pH 7.4. |
plastic transfer pipette | Medi'Ray | RL200C | |
poloxamer 188 | BASF Corporation | ||
pressure injector | Applied Scientific Instruments | MPPI-2 | |
rotary evaporator | Büchi, Flawil, Switzerland | Büchi R-215 Rotavapor | |
Scanning confocal microscope | Olympus | Olympus FV1000 FluoView | |
Sorvall WX+ Ultracentrifuge | Thermo Scientific | 75000090 | |
stereomicroscope | Leica | MZ12 | |
Tricaine | Sigma-Aldrich | A5040-25G | Make 4 mg/mL stock solution (in deionzed H2O) and keep at -20 °C. |
triton-X100 | Sigma-Aldrich | X100-100ML | |
Ultrasonic bath | Thermo Scientific | FB-11205 | |
Volocity Image Analysis Software | PerkinElmer | version 6.3 | |
water bath | |||
Zetasizer Nano | Malvern Instruments Ltd | Zetasizer Nano ZS ZEN 3600 |