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

This protocol facilitates screening of new formulations to better understand how modifications affect their tissue residence times, which is critical in designing an optimal drug delivery system. This technique is noninvasive and allows realtime imaging, which reduces the excessive use of animals for sampling. Drug delivery systems can be redesigned for imaging and therapeutic applications by simply including the therapeutic drug and the imaging moiety in one nano-carrier.

This has useful applications in both treating cancer and infectious disease. Demonstrating the procedure will be Rasha Msallam, a senior research fellow from my laboratory. To begin with, two hours before subconjunctival injection, inject the mouse intravenously via tail with 2.5 milligrams per kilogram of Evans blue, or EB.After sedating the mouse with 5%isoflurane, transfer the mouse to a nose cone and maintain sedated mouse on a heating pad.

Then, trim the whiskers near the eye to be injected. Instill a drop of 0.5%proxymetacaine hydrochloride solution directly on the eye. Next, load a 10-microliter glass syringe equipped with a 32-gauge needle with fluorescent liposomes and ensure that the air bubbles are not present in the syringe.

Using a tweezer, lift the conjunctiva and slowly inject liposomes into the subconjunctival space, then withdraw the needle slowly, preventing back flow. Ensure the formation of bleb filled with fluorescent liposomes. Administer a 1%fusitic acid drop on the eye and monitor the mouse until it regains consciousness.

Switch on the CLM system. Connect the probe to the CLM system. Choose the field-of-view and the location for the acquisition files at this point.

Adjust the laser intensity to ensure that the fluorescence detection for fluorescein DHPE is in the linear range, and keep the intensity consistent for comparison between images taken at different time points. After the system is warmed up for 15 minutes, calibrate the system according to the manufacturer's instructions using the calibration kit containing the cleansing, rinsing, and fluoro-4 solutions for each laser. Briefly, start calibrating by immersing the tip for five seconds each in the cleansing vial, and then in the rinsing vial.

Leave the tip in the air for background recording to normalize the background values from the probes different fibers, ensuring image uniformity. Again, immerse the tip for five seconds in the cleansing vial, and then in the rinsing vial. Next, immerse the tip in the vial containing fluoro-4 488-nanometer for five seconds to normalize the signal values.

Repeat the tip immersion for five seconds in the cleansing vial followed by the rinsing vial. Keep the tip in the rinsing vial until the fluorescence signal recorded in the previous step disappears. Then, transfer the tip in the vial containing fluoro-4 660-nanometer to normalize signal values from different fibers in the probe.

After calibration, ensure the value is less than 100 for the probe's background. Otherwise, perform repeated cleaning of the probe with a cotton tip applicator. Switch on the animal temperature controller with an attached heating pad, and adjust the temperature to 37 degrees Celsius.

After covering the heating pad with a surgical drape, fix the nose cone on the heating pad. After sedating the mouse using 5%isoflurane in an induction chamber, transfer the mouse to the nose cone and maintain the sedated mouse on a heating pad. Then, instill a drop of anesthetic 0.5%proxymetacaine hydrochloride solution onto the eye.

To wash the ocular surface and avoid the crystallization of the lens, keep the eyes lubricated by dropping a few saline drops. Then, rotate and adjust the eyepiece of the microscope to view the mouse eye ergonomically in the direct focus at 0.67 times magnification. Turn on the laser and place the probe like a pen directly on the eye region to be imaged.

Then, start recording acquisition to observe the fluorescence in the eye at the region of interest. Stop the recording when all regions are flagged and labeled according to the eye map to know the exact probe location at the exact frame. The acquisition files will be saved automatically as video files in the location previously chosen.

The fluorescence imaging revealed distinct differentiation between the sclera and cornea regions. High vascularization of the episclera and conjunctiva caused the sclera region to stain red with EB.The cornea, which lacks vasculature, appeared black. The contrast fluorescence of limbus and sclera was observed in seven-day study.

Fluorescein dye-injected control mice confirmed the specificity of liposome-derived fluorescence. The fluorescence intensities were high on first and third days. Green fluorescence proxied the liposome reduction in the limbus and sclera regions over time.

The differences in the fluorescence signals from post-injection day one to day three at temporal limbus and superior limbus regions were not significant, indicating preference of neutral liposomes to the limbus region, particularly the corneal periphery. For the temporal and superior regions on the post-injection day one, fluorescence in the sclera was six times higher than in the limbus. By the third and seventh day, liposomes were significantly reduced in the sclera in the temporal and superior regions, attributed to ocular clearance mechanisms.

The lowest amount of fluorescence was detected in the nasal and inferior regions, due to the distance from the injection site. It is important to label the fleck frames according to the eye map before the recording is stopped. Keep the laser intensity consistent, and define the maximum background value for proper comparison across different experiments.

Following the identification of lead formulations, comprehensive pharmacokinetic and biodistribution studies in animals can be conducted to study the drug's bioavailability, and validate the therapeutic efficacy of their formulations.