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 ...

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