Spatio-Temporal In Vivo Imaging of Ocular Drug Delivery Systems using Fiberoptic Confocal Laser Microendoscopy

Summary

We present a protocol for the use of fiberoptic confocal laser microendoscopy (CLM) to non-invasively study the spatio-temporal distribution of liposomes in the eye after subconjunctival injection.

Abstract

Subconjunctival injection is an attractive route to administer ocular drugs due to easy trans-scleral access that bypasses anterior ocular barriers, such as the cornea and conjunctiva. While therapeutic effects and pharmacokinetics of the drugs upon subconjunctival injection have been described in some studies, very few assess the ocular distribution of drugs or drug delivery systems (DDS). The latter is critical for the optimization of intraocular DDS design and drug bioavailability to achieve the desired ocular localization and duration of action (e.g., acute versus. prolonged). This study establishes the use of fiberoptic confocal laser microendoscopy (CLM) to qualitatively study the ocular distribution of fluorescent liposomes in real-time in live mice after sub-conjunctival injection. Being designed for in vivo visual inspection of tissues at the microscopic level, this is also the first full description of the CLM imaging method to study spatio-temporal distribution of injectables in the eye after subconjunctival injection.

Introduction

The blood clearance, tissue distribution, and target occupancy of drugs in living systems are pillars to understanding in vivo drug disposition. In preclinical animal models, these parameters are typically assessed by frequent blood and tissue sampling at particular time points post drug administration. However, these procedures are generally invasive, often include non-survival measurements, and necessitate large animal cohorts for statistical powering. There might be extra cost and time incurred, along with ethical concerns for excessive use of animals. As a result, non-invasive imaging is fast becoming an integral step in biodistributions studies. Confocal laser microendoscopy (CLM^1,2) is well-suited for ocular applications to non-invasively image the spatio-temporal distribution of therapeutics in the eyes of live animals with high sensitivity and high resolution^1,3,4.

CLM has the potential to facilitate robust screening of ocular drug delivery systems (DDS), such as liposomes, prior to comprehensive quantification of the DDS and drug bioavailability. Liposomes are attractive for their flexibility in tuning their physicochemical and biophysical properties^{5,6,7,8,9,10,11} to encapsulate a large variety of therapeutic cargo and control the tissue site of drug release and duration of action. Liposomes have been used in ocular applications for the delivery of large molecules, such as the monoclonal antibody bevacizumab¹², and small molecules like cyclosporine¹³ and ganciclovir¹⁴. Drug-loaded liposomes have longer biological half-lives and prolonged therapeutic effects compared to non-liposomal "free drug" formulations. However, drug distribution in ocular tissue is typically extrapolated from drug concentrations in fluid components of the eye (i.e., blood, aqueous humor, and vitreous humor^15,16,17). As the initial in vivo fate of the loaded drug cargo is defined by the properties of the nanocarrier itself, CLM imaging of the fluorescent liposomes can serve as a surrogate for the drug to reveal tissue targeting and in situ tissue residence times. Furthermore, visual evidence of delivery with CLM can steer DDS re-design, evaluate therapeutic benefits of the drug, and perhaps even predict adverse biological events (e.g., tissue toxicity due to undesirable localization of DDS for protracted periods of time).

Herein, a step-by-step procedure is detailed on how to study the ocular biodistribution of liposomes in live mice with a dual-band CLM system. This specific CLM system can detect two-color fluorescence (with green and red excitation lasers at 488 nm and 660 nm) in real-time, with a frequency of 8 frames/s. By physically placing the detection probe on the eye, the protocol demonstrates image acquisition and analysis of green-fluorescent liposomes upon subconjunctival administration in mice pre-injected intravenously (IV) with 2% Evans Blue (EB) dye. EB dye helps visualize the vascularized structures in the red fluorescence channel. We show representative results from a study assessing 100 nm neutral liposomes composed of the phospholipid POPC (i.e., 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine) and doped with fluorescein-tagged phospholipid Fl-DHPE (i.e., N-(fluorescein-5-thiocarbamoyl)-1,2-dihexa-decanoylsn-glycero-3-phosphoethanolamine) at a ratio of 95% POPC: 5% Fl-DHPE (Figure 1B). CLM is able to capture the green fluorescein-tagged liposomes at 15 µm axial and 3.30 µm lateral resolution by delineation of EB-stained ocular tissue boundaries.

Protocol

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) at SingHealth (Singapore). Female C57BL/6 J mice (6- 8 weeks old; 18-20 g) were obtained from InVivos, Singapore, and housed in a temperature and light-controlled vivarium of Duke-NUS Medical School, Singapore. Animals were treated in accordance with the guidelines from the Association for Research in Vision and Ophthalmology (ARVO) statement for the use of animals in ophthalmic and vision research.

NOTE: A flow chart highlighting the main procedures is shown in Figure 2.

1. Preparation of contrast agents: Evans Blue (EB) and liposomes

1. For 2% EB dye solution, dissolve 1 g of EB in 50 mL of sterile saline. Filter the solution using 0.22 µm filters into 1.5 mL sterile tubes and store them at room temperature for later use.
2. For green-fluorescent liposomes, add POPC/Fl-DHPE (95:5), chloroform/methanol (2:1) into a 100 mL round bottom flask. Use a rotary evaporator at 150 rpm at 40 °C for 1 h, with vacuum maintained at 0 mbar¹ to create a thin lipid film.
   NOTE: When comparing effects of liposomal properties (e.g., size, charge, lipid saturation, lipid chain length) on distribution, maintain a fixed percentage of Fl-DHPE or other fluorescent lipids to confirm that the results observed are due to the effect of the properties tested, and not to the variable load of large hydrophobic dyes.
3. Hydrate the lipid film with phosphate-buffered saline (to achieve 26.3 mM of fluorescent liposomes) at 40 °C to form multi-lamellar vesicles (MLV). Load the MLVs into a glass syringe for manual extrusion (30 times using a 0.08 µm pore size polycarbonate filter) to achieve the desired size of 100 nm.
   NOTE: The temperature for hydration must be higher than the transition temperature of the lipids.
4. Filter the liposomes by passing them through a 0.22 µm sterile syringe filter. Confirm the hydrodynamic diameter (D_H) of the liposomes using a dynamic light scattering system.

2. Administration of EB and liposomes in live mice

1. Inject mouse with EB IV (intravenous) via the tail vein (2.5 mg/kg), 2 h before subconjunctival injection.
2. For subconjunctival injection, first, sedate the mouse using 5% isoflurane via inhalation in an induction chamber to achieve an adequate plane of anesthesia. Transfer the mouse to a nose cone and maintain sedation at 2%-2.5% of isoflurane while on a heating pad throughout the procedure.
3. Trim the whiskers near the eye to be injected and instill a drop of topical anesthetic 0.5% proxymetacaine hydrochloride solution directly on the eye.
4. Load a 10 µL glass syringe (with 32 G needle) with fluorescent liposomes (Fl-DHPE: 0.78 mg/kg) and dispel all air bubbles in the syringe prior to injection.
   NOTE: Up to 20 µL of injectate can be accommodated in the subconjunctival space of mice^18,19.
5. Using a tweezer, lift the conjunctiva slightly and inject slowly into the subconjunctival space (Figure 1A). Withdraw the needle slowly to prevent backflow. Ensure that a visible bleb filled with fluorescent liposomes is formed (Figure 1C).
6. Administer a drop of antibiotic 1% fusidic acid on the eye after the injection and monitor the mouse until it regains consciousness.

3. CLM set-up

1. Switch on the CLM system and make sure both the connector and the distal tip of the scanning probe are clean.
2. Clean the connector of the scanning probe using an optical connector cleaner by following the manufacturer's instructions.
   1. Press the rachet (often colored) of the optical connector cleaner to reveal a cleaning ribbon.
   2. Position the connector in contact with the cleaning ribbon and slide the connector along the ribbon while maintaining contact.
3. Clean the distal tip (also known as the scanning tip) of the probe by dipping it into the cleansing solution, followed by the rinsing solution provided by the manufacturer. A cotton tip applicator can also be used for more thorough cleaning if the tip is very dirty.
4. Connect the probe to the CLM system. Choose the Field of view (FOV) and the location for the acquisition files at this point.
   NOTE: Adjust the laser intensity at this step to ensure that fluorescence detection for Fl-DHPE is in the linear range. Laser intensity is to be kept consistent for comparison between images taken at different time points.
5. Allow the system to warm up for 15 min as instructed and use the calibration kit to calibrate the system according to the manufacturer's instructions.
   NOTE: In the calibration kit, there are three vials for each laser containing the following solutions: cleansing solution, rinsing solution, and fluorophore 488/660 nm solution for internal calibration. The calibration steps are prompted by the system and are to be followed accordingly.
   1. Immerse the tip in the cleansing vial followed by the rinsing vial (5 s in each vial). Leave it in the air for background recording for both channels.
      NOTE: This step is very crucial as it normalizes the background values from different fibers of the probe and ensures image uniformity.
   2. Immerse the tip in the cleansing vial followed by the rinsing vial (5 s in each vial). Immerse the tip in fluorophore 488 nm vial for 5 seconds to normalize signal values from different fibers in the probe.
   3. Immerse the tip in the cleansing vial followed by the rinsing vial (5 s in each vial). Immerse the tip in the rinsing vial until the fluorescence signal recorded in 3.5.2. disappears. Immerse the tip in fluorophore 660 nm vial to normalize signal values from different fibers in the probe.
      NOTE: Follow all the calibration steps in order to achieve proper calibration and optimal image quality.
6. After calibrating the probe, check to make sure the background values for the probes are as low as possible. For the CLM system used, keep the background values below 100. Perform repeated cleaning of the probe with a cotton tip applicator and calibration if values are above 100/defined user value or if the probe appears to be dirty. This is to ensure that the background noise is kept around the same value.
   NOTE: It is important to define the maximum background value (for example, 100 as stated in step 3.6) to ensure that probe conditions are similar. This will allow proper quantitative comparison between images taken at different time points. The value may differ in different systems and probe conditions.
7. Switch on the animal temperature controller (ATC). Adjust the ATC to 37 °C. Cover the heating pad with a surgical drape and fix the nose cone on the heating pad.
   NOTE: ATC with an attached heating pad is required to ensure that the animal is kept warm throughout the imaging duration.
8. Clamp the stand of the dissecting microscope to the tabletop to secure it. Rotate and adjust the eyepiece of the microscope to view the mouse eye ergonomically through the eyepiece when the user is seated (make the adjustments after placing the animal).
9. Sedate the mouse using 5% isoflurane in an induction chamber. Transfer the mouse to the nose cone once the animal is non-responsive and maintain sedation at 2%-2.5% isoflurane while on a heating pad throughout the procedure.
10. Trim the whiskers of the mouse and instill a drop of anesthetic 0.5% proxymetacaine hydrochloride solution onto the eye.
11. To make sure the eyes are clean, drop a few drops of saline to wash the ocular surface.
12. Adjust the microscope so that the mouse eye is in direct focus at 0.67x magnification.
    NOTE: Make sure to lubricate the eyes with saline. If the eyes are not lubricated throughout the imaging session, they can get dry, causing the lens to crystallize. As a result, during CLM imaging, the lens can emit a background red fluorescence.

4. Live imaging of mouse eyes with CLM and acquisition

1. Turn on the laser, place the probe on the eye and start recording acquisition to observe the fluorescence in the eye at the regions indicated on the eye map in Figure 3.
   NOTE: Hold the probe like a pen with the distal end of the probe directly onto the region to be imaged.
2. Stop the recording when all regions have been flagged and labeled. The acquisition files will be saved automatically in the file location chosen in step 3.4.
   NOTE: The file will be saved as a video file which can be exported to individual images. Label the flag according to the eye map to know exactly the location of the probe at the exact frame the recording was done.

5. Image analysis

1. Using the same CLM acquisition software, export the image acquisition files for further analysis. Click on File | Export and choose the format to be exported to. Mkt format files will allow adjustments of look up table (LUT) and further exports to image file formats using the CLM viewer software.
2. For accurate comparison of fluorescence intensity, use the same LUT adjusted for each channel when exporting all image files.
   NOTE: Choose the minimum and maximum LUT threshold with respect to those of the control mouse (with no liposomes injected) to minimize background fluorescence readings.
3. Open the image in an appropriate image processing software/freeware program (e.g., ImageJ). Draw the region of interest (ROI).
   NOTE: ROI here refers to the region of interest in the processing program. In most cases, ROI will be the whole image scanned. However, in the case of imaging the limbus, the probe cannot just get the image of the limbus separately. Therefore, an ROI has to be drawn to 'quantify' the fluorescence in the limbus region, as drawn in Figure 4.To keep the ROI consistent, use the same ROI across all images.
4. Measure and record the ROI values for green fluorescence. Enter the values in a spreadsheet. Tabulate the average and fluorescence intensity (a.u.) values of the ROI.

6. Histology assessment

1. Euthanize the mouse using a method approved by the local IACUC. 
2. Enucleate the eye and fix the eye in 1 mL of 4% formaldehyde or 10% formalin solution overnight.
3. Trim excess fats and embed the eye in the Optimal Cutting Temperature (OCT) compound and keep it frozen in a -80 °C freezer for at least one day.
4. Cut sections of 5 µm thickness in the cryostat with cutting temperature maintained at 20 °C. Transfer the section to a Poly-L-Lysine-coated microscope slide.
   NOTE: Histology can serve as additional validation of the distribution of DDS. However, it requires additional optimization, technical expertise, and sacrifice of animals which the study aims to reduce by using CLM.

Results

The protocol demonstrates the utility of CLM to assess the spatio-temporal ocular distribution of green fluorescent liposomes administered through subconjunctival injection. To make use of the dual-color capability (488 nm and 660 nm excitation wavelengths) of the CLM system, 100 nm neutral POPC liposomes to be injected were doped with 5% Fl-DHPE (composition and characterization data are shown in Figure 1B), and EB was injected IV to identify landmarks in the eye. The presence of a thin lay...

Discussion

As shown from the results, CLM provides a simple and feasible method to image the ocular distribution of liposomes in the eye. We previously demonstrated the use of CLM to characterize the localization of various liposomal formulations within the mouse eye over time¹. For non-invasive applications, CLM permits real-time imaging of the anterior ocular surface for insights on how liposomes are distributed in the eye from the same animal. This makes CLM suitable to pre-screen nanocarrier/DDS prior to...

Materials

Name	Company	Catalog Number	Comments
0.08 µm polycarbonate filter	Whatman, USA	110604
0.22 µm syringe filter	Fisherbrand, Ireland	09-720-3
0.5% Proxymetacaine hydrochloride sterile opthalmic solution	Alcon, Singapore
10 µL Glass Syringe	Hamilton, USA	65460-06
1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)	Avanti, USA	850457
32 G needle (Hamilton, 0.5” PT4)	Hamilton, USA	7803-04
Animal Temperature Controller with heating plate (15 cm x 20 cm)	WPI, USA	ATC 2000 & 61800
Cellvizio Dual Band, S1500 Probe and Quantikit (Calibration kit in step 3.5)	Mauna Kea Technologies, France		Tip diameter: 1.5 mm, field of view: 600 µm x 500 µm, axial resolution: 15 µm, lateral resolution: 3.3 µm
Chloroform	Sigma Aldrich, USA	472476
Dumont Tweezers #5, Dumostar	WPI, USA	500233	11 cm, Straight, 0.1 mm x 0.06 mm Tips
Evans Blue	Sigma Aldrich, USA	E2129
Fusidic acid eye drop	LEO Pharma, Denmark
ImageJ	National Institutes of Health, USA		https://imagej.nih.gov/ij/
Isoflurane	Piramal, USA
Malvern Zetasizer Nano ZS	Malvern Panalytical, UK
Methanol	Sigma Aldrich, USA	179337
Mini Extruder	Avanti, USA	610020
N-(fluorescein-5-thiocarbamoyl)-1,2-dihexadecanoylsn-glycero-3-phosphoethanolamine (triethylammonium salt) (FL-DHPE)	Invitrogen, USA	F362
Phosphate Buffered Saline	Gibco, USA	10010023
Stereomicroscope System with table clamp stand	Olympus, Tokyo, Japan	SZ51 & SZ2-STU3