Name: spatio-temporal in vivo imaging of ocular drug delivery systems using fiberoptic confocal laser microendoscopy
Uploaded: 2021-09-27T14:00:00
Description: Watch this Scientific Journal Video about Spatio-Temporal In Vivo Imaging of Ocular Drug Delivery Systems using Fiberoptic Confocal Laser Microendoscopy at JoVE.com

This research was funded by NTU-Northwestern Institute for Nanomedicine (NNIN) grant awarded (to SV) and in part by Singapore National Research Foundation Grant AG/CIV/GC70-C/NRF/2013/2 and Singapore&#8217;s Health and Biomedical Sciences (HBMS) Industry Alignment Fund Pre-Positioning (IAF-PP) grant H18/01/a0/018 administered by the Agency for Science, Technology and Research (A*STAR) (to AMC). Thanks to members from Duke-NUS Laboratory for Translational and Molecular Imaging (LTMI) for facilitating the logistics and execution of the studies and training on equipment. Special thanks to Ms. Wisna Novera for her editorial assistance.

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.
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<ol>
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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...

The blood clearance, tissue distribution, and target occupancy of drugs in living systems are pillars to understanding in vivo drug&#160;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 (CLM1,2) 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 resolution1,3,4.
CLM has the potential to facilitate robust screening of ocular drug delivery systems (DDS), such as&#160;liposomes, prior to comprehensive quantification of the DDS and drug bioavailability. Liposomes are attractive for their flexibility in tuning their physicochemical and biophysical properties5,6,7,8,9,10,11 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 &#34;free drug&#34; formulations.&#160;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 humor15,16,17). 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 &#181;m axial and 3.30 &#181;m lateral resolution by delineation of EB-stained ocular tissue boundaries.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.08 &#181;m polycarbonate filter</td>
			<td>Whatman, USA</td>
			<td>110604</td>
			<td></td>
		</tr>
		<tr>
			<td>0.22 &#181;m syringe filter</td>
			<td>Fisherbrand, Ireland</td>
			<td>09-720-3</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5% Proxymetacaine hydrochloride sterile opthalmic solution</td>
			<td>Alcon, Singapore</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10 &#181;L Glass Syringe</td>
			<td>Hamilton, USA</td>
			<td>65460-06</td>
			<td></td>
		</tr>
		<tr>
			<td>1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)</td>
			<td>Avanti, USA</td>
			<td>850457</td>
			<td></td>
		</tr>
		<tr>
			<td>32 G needle (Hamilton, 0.5&#8221; PT4)</td>
			<td>Hamilton, USA</td>
			<td>7803-04</td>
			<td></td>
		</tr>
		<tr>
			<td>Animal Temperature Controller with heating plate (15 cm x 20 cm)</td>
			<td>WPI, USA</td>
			<td>ATC 2000 &#38; 61800</td>
			<td></td>
		</tr>
		<tr>
			<td>Cellvizio Dual Band, S1500 Probe and Quantikit (Calibration kit in step 3.5)</td>
			<td>Mauna Kea Technologies, France</td>
			<td></td>
			<td>Tip diameter: 1.5 mm, field of view: 600 &#181;m x 500 &#181;m, axial resolution: 15 &#181;m, lateral resolution: 3.3 &#181;m</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Sigma Aldrich, USA</td>
			<td>472476</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont Tweezers #5, Dumostar</td>
			<td>WPI, USA</td>
			<td>500233</td>
			<td>11 cm, Straight, 0.1 mm x 0.06 mm Tips</td>
		</tr>
		<tr>
			<td>Evans Blue</td>
			<td>Sigma Aldrich, USA</td>
			<td>E2129</td>
			<td></td>
		</tr>
		<tr>
			<td>Fusidic acid eye drop</td>
			<td>LEO Pharma, Denmark</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>National Institutes of Health, USA</td>
			<td></td>
			<td>https://imagej.nih.gov/ij/</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Piramal, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Malvern Zetasizer Nano ZS</td>
			<td>Malvern Panalytical, UK</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma Aldrich, USA</td>
			<td>179337</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini Extruder</td>
			<td>Avanti, USA</td>
			<td>610020</td>
			<td></td>
		</tr>
		<tr>
			<td>N-(fluorescein-5-thiocarbamoyl)-1,2-dihexadecanoylsn-glycero-3-phosphoethanolamine (triethylammonium salt) (FL-DHPE)</td>
			<td>Invitrogen, USA</td>
			<td>F362</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Gibco, USA</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope System with table clamp stand</td>
			<td>Olympus, Tokyo, Japan</td>
			<td>SZ51 &#38; SZ2-STU3</td>
			<td></td>
		</tr>
	</tbody>
</table>

spatio-temporal in vivo imaging of ocular drug delivery systems using fiberoptic confocal laser microendoscopy

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.

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...

Watch this Scientific Journal Video about Spatio-Temporal In Vivo Imaging of Ocular Drug Delivery Systems using Fiberoptic Confocal Laser Microendoscopy at JoVE.com

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

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.

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

Subconjunctival injection is an attractive route to administer ocular drugs due to easy trans-scleral access that bypasses anterior ocular barriers, such ...

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.

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Sub-micrometer carriers (nanocarriers; NCs) enhance efficacy of drugs by improving solubility, stability, circulation time, targeting, and release. ...

A Label-free Technique for the Spatio-temporal Imaging of Single Cell Secretions

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning ...

Multimodal Imaging and Spectroscopy Fiber-bundle Microendoscopy Platform for Non-invasive, In Vivo Tissue Analysis

Multimodal Imaging and Spectroscopy Fiber-bundle Microendoscopy Platform for Non-invasive, In Vivo Tissue Analysis

Recent fiber-bundle microendoscopy techniques enable non-invasive analysis of in vivo tissue using either imaging techniques or a combination of ...

Manufacture and Drug Delivery Applications of Silk Nanoparticles

Silk is a promising biopolymer for biomedical and pharmaceutical applications due to its outstanding mechanical properties, biocompatibility and ...

Comprehensive Evaluation of the Effectiveness and Safety of Placenta-Targeted Drug Delivery Using Three Complementary Methods

No effective treatments currently exist for placenta-associated pregnancy complications, and developing strategies for the targeted delivery of drugs to ...

Preparing Protein Producing Synthetic Cells using Cell Free Bacterial Extracts, Liposomes and Emulsion Transfer

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell ...