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>
	<li>Chaw, S. Y., Novera, W., Chacko, A. -. M., Wong, T. T. L., Venkatraman, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+fate+of+liposomes+after+subconjunctival+ocular+delivery.">In vivo fate of liposomes after subconjunctival ocular delivery.</a> Journal of Controlled Release. 329, 162-174 (2021).</li><li>Kuo, J. C. -. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+colorectal+dysplasia+using+fluorescently+labelled+lectins.">Detection of colorectal dysplasia using fluorescently labelled lectins.</a> Scientific Reports. 6 (1), 24231 (2016).</li><li>Wu, Y. -. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+custom+multiphoton+microscopy+platform+for+live+imaging+of+mouse+cornea+and+conjunctiva.">A custom multiphoton microscopy platform for live imaging of mouse cornea and conjunctiva.</a> Journal of Visualized Experiments: JoVE. (159), e60944 (2020).</li><li>Zhivov, A., Stachs, O., Kraak, R., Stave, J., Guthoff, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+confocal+microscopy+of+the+ocular+surface.">In vivo confocal microscopy of the ocular surface.</a> The Ocular Surface. 4 (2), 81-93 (2006).</li><li>Bassyouni, F., ElHalwany, N., Ab del Rehim, M., Neyfeh, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+and+new+technologies+applied+in+controlled+drug+delivery+system.">Advances and new technologies applied in controlled drug delivery system.</a> Research on Chemical Intermediates. 41 (4), 2165-2200 (2015).</li><li>Sercombe, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+and+challenges+of+liposome+assisted+drug+delivery.">Advances and challenges of liposome assisted drug delivery.</a> Frontiers in Pharmacology. 6, (2015).</li><li>Koning, G. A., Storm, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+drug+delivery+systems+for+the+intracellular+delivery+of+macromolecular+drugs.">Targeted drug delivery systems for the intracellular delivery of macromolecular drugs.</a> Drug Discovery Today. 8 (11), 482-483 (2003).</li><li>Metselaar, J. M., Storm, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liposomes+in+the+treatment+of+inflammatory+disorders.">Liposomes in the treatment of inflammatory disorders.</a> Expert Opinion on Drug Delivery. 2 (3), 465-476 (2005).</li><li>Ding, B. S., Dziubla, T., Shuvaev, V. V., Muro, S., Muzykantov, V. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+drug+delivery+systems+that+target+the+vascular+endothelium.">Advanced drug delivery systems that target the vascular endothelium.</a> Molecular Interventions. 6 (2), 98-112 (2006).</li><li>Hua, S., Wu, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+lipid-based+nanocarriers+for+targeted+pain+therapies.">The use of lipid-based nanocarriers for targeted pain therapies.</a> Frontiers in Pharmacology. 4, 143 (2013).</li><li>Sharma, A., Sharma, U. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liposomes+in+drug+delivery:+Progress+and+limitations.">Liposomes in drug delivery: Progress and limitations.</a> International Journal of Pharmaceutics. 154 (2), 123-140 (1997).</li><li>Abrishami, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation,+characterization,+and+in+vivo+evaluation+of+nanoliposomes-encapsulated+Bevacizumab+(Avastin)+for+intravitreal+administration.">Preparation, characterization, and in vivo evaluation of nanoliposomes-encapsulated Bevacizumab (Avastin) for intravitreal administration.</a> Retina. 29 (5), 699-703 (2009).</li><li>Pleyer, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ocular+absorption+of+cyclosporine+A+from+liposomes+incorporated+into+collagen+shields.">Ocular absorption of cyclosporine A from liposomes incorporated into collagen shields.</a> Current Eye Research. 13 (3), 177-181 (1994).</li><li>Shen, Y., Tu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+ocular+pharmacokinetics+of+ganciclovir+liposomes.">Preparation and ocular pharmacokinetics of ganciclovir liposomes.</a> The AAPS Journal. 9 (3), 371-377 (2007).</li><li>Weijtens, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+concentration+of+dexamethasone+in+aqueous+and+vitreous+after+subconjunctival+injection.">High concentration of dexamethasone in aqueous and vitreous after subconjunctival injection.</a> American Journal of Ophthalmology. 128 (2), 192-197 (1999).</li><li>Voss, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+novel+injectable+drug+delivery+system+for+subconjunctival+glaucoma+treatment.">Development of a novel injectable drug delivery system for subconjunctival glaucoma treatment.</a> Journal of Controlled Release. 214, 1-11 (2015).</li><li>Giarmoukakis, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biodegradable+nanoparticles+for+controlled+subconjunctival+delivery+of+latanoprost+acid:+In+vitro+and+in+vivo+evaluation.+Preliminary+results.">Biodegradable nanoparticles for controlled subconjunctival delivery of latanoprost acid: In vitro and in vivo evaluation. Preliminary results.</a> Experimental Eye Research. 112, 29-36 (2013).</li><li>Shah, N. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravitreal+and+subconjunctival+melphalan+for+retinoblastoma+in+transgenic+mice.">Intravitreal and subconjunctival melphalan for retinoblastoma in transgenic mice.</a> Journal of Ophthalmology. 2014, 829879 (2014).</li><li>Dastjerdi, M. H., Sadrai, Z., Saban, D. R., Zhang, Q., Dana, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+Penetration+of+Topical+and+Subconjunctival+Bevacizumab.">Corneal Penetration of Topical and Subconjunctival Bevacizumab.</a> Investigative ophthalmology &amp; visual science. 52 (12), 8718-8723 (2011).</li><li>Ezra-Elia, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Can+an+in+vivo+imaging+system+be+used+to+determine+localization+and+biodistribution+of+AAV5-mediated+gene+expression+following+subretinal+and+intravitreal+delivery+in+mice.">Can an in vivo imaging system be used to determine localization and biodistribution of AAV5-mediated gene expression following subretinal and intravitreal delivery in mice.</a> Experimental Eye Research. 176, 227-234 (2018).</li><li>Movila, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+endoscopic+technology+for+real-time+monitoring+of+inflammation+caused+in+experimental+periodontitis.">Intravital endoscopic technology for real-time monitoring of inflammation caused in experimental periodontitis.</a> Journal of Immunological Methods. 457, 26-29 (2018).</li><li>Vanherp, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bronchoscopic+fibered+confocal+fluorescence+microscopy+for+longitudinal+in+vivo+assessment+of+pulmonary+fungal+infections+in+free-breathing+mice.">Bronchoscopic fibered confocal fluorescence microscopy for longitudinal in vivo assessment of pulmonary fungal infections in free-breathing mice.</a> Scientific Reports. 8 (1), 3009 (2018).</li><li>Chagnon, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+intravital+endoscopic+confocal+fluorescence+microscopy+of+normal+and+acutely+injured+rat+lungs.">In vivo intravital endoscopic confocal fluorescence microscopy of normal and acutely injured rat lungs.</a> Laboratory Investigation. 90 (6), 824-834 (2010).</li><li>Yun, J. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+near-infrared+fluorescence+conjugation+on+the+anti-cancer+potential+of+cetuximab.">The effect of near-infrared fluorescence conjugation on the anti-cancer potential of cetuximab.</a> Laboratory Animal Research. 34 (1), 30-36 (2018).</li></ol>

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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Research

JoVE Journal

Bioengineering

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution. Traditionally, multiple experiments are run in parallel, with individual samples removed from the study at sequential time points for evaluation by light microscopy. Several intravital techniques have been developed, with confocal, multiphoton, and second harmonic microscopy all demonstrating their ability to be used for imaging in situ 1. With these systems, however, the required infrastructure is complex and expensive, involving scanning laser systems and complex light sources. Here we present a protocol for the design and assembly of a high-resolution microendoscope which can be built in a day using off-the-shelf components for under US$5,000. The platform offers flexibility in terms of image resolution, field-of-view, and operating wavelength, and we describe how these parameters can be easily modified to meet the specific needs of the end user.
We and others have explored the use of the high-resolution microendoscope (HRME) in in vitro cell culture 2-5, in excised 6 and living animal tissues 2,5, and in human tissues in vivo 2,7. Users have reported the use of several different fluorescent contrast agents, including proflavine 2-4, benzoporphyrin-derivative monoacid ring A (BPD-MA) 5, and fluoroscein 6,7, all of which have received full, or investigational approval from the FDA for use in human subjects. High-resolution microendoscopy, in the form described here, may appeal to a wide range of researchers working in the basic and clinical sciences. The technique offers an effective and economical approach which complements traditional benchtop microscopy, by enabling the user to perform high-resolution, longitudinal imaging in situ.

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

In many biological and clinical situations it is advantageous to study cellular processes as they evolve in their native microenvironment. Here we describe the assembly and use of a low-cost fiber-optic microscope which can provide real time imaging in cell culture, animal studies, and clinical patient studies.

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution.  ...

Video-rate Scanning Confocal Microscopy and Microendoscopy

Confocal microscopy has become an invaluable tool in biology and the biomedical sciences, enabling rapid, high-sensitivity, and high-resolution optical sectioning of complex systems. Confocal microscopy is routinely used, for example, to study specific cellular targets1, monitor dynamics in living cells2-4, and visualize the three dimensional evolution of entire organisms5,6. Extensions of confocal imaging systems, such as confocal microendoscopes, allow for high-resolution imaging in vivo7 and are currently being applied to disease imaging and diagnosis in clinical settings8,9.
Confocal microscopy provides three-dimensional resolution by creating so-called "optical sections" using straightforward geometrical optics. In a standard wide-field microscope, fluorescence generated from a sample is collected by an objective lens and relayed directly to a detector. While acceptable for imaging thin samples, thick samples become blurred by fluorescence generated above and below the objective focal plane. In contrast, confocal microscopy enables virtual, optical sectioning of samples, rejecting out-of-focus light to build high resolution three-dimensional representations of samples.
Confocal microscopes achieve this feat by using a confocal aperture in the detection beam path. The fluorescence collected from a sample by the objective is relayed back through the scanning mirrors and through the primary dichroic mirror, a mirror carefully selected to reflect shorter wavelengths such as the laser excitation beam while passing the longer, Stokes-shifted fluorescence emission. This long-wavelength fluorescence signal is then passed to a pair of lenses on either side of a pinhole that is positioned at a plane exactly conjugate with the focal plane of the objective lens. Photons collected from the focal volume of the object are collimated by the objective lens and are focused by the confocal lenses through the pinhole. Fluorescence generated above or below the focal plane will therefore not be collimated properly, and will not pass through the confocal pinhole1, creating an optical section in which only light from the microscope focus is visible. (Fig 1). Thus the pinhole effectively acts as a virtual aperture in the focal plane, confining the detected emission to only one limited spatial location.
Modern commercial confocal microscopes offer users fully automated operation, making formerly complex imaging procedures relatively straightforward and accessible. Despite the flexibility and power of these systems, commercial confocal microscopes are not well suited for all confocal imaging tasks, such as many in vivo imaging applications. Without the ability to create customized imaging systems to meet their needs, important experiments can remain out of reach to many scientists.
In this article, we provide a step-by-step method for the complete construction of a custom, video-rate confocal imaging system from basic components. The upright microscope will be constructed using a resonant galvanometric mirror to provide the fast scanning axis, while a standard speed resonant galvanometric mirror will scan the slow axis. To create a precise scanned beam in the objective lens focus, these mirrors will be positioned at the so-called telecentric planes using four relay lenses. Confocal detection will be accomplished using a standard, off-the-shelf photomultiplier tube (PMT), and the images will be captured and displayed using a Matrox framegrabber card and the included software.

The complete construction of a custom, real-time confocal scanning imaging system is described. This system, which can be readily used for video-rate microscopy and microendoscopy, allows for an array of imaging geometries and applications not accessible using standard commercial confocal systems, at a fraction of the cost.

Confocal microscopy has become an invaluable tool in biology and the biomedical sciences, enabling rapid, high-sensitivity, and high-resolution optical ...

Spatio-Temporal Manipulation of Small GTPase Activity at Subcellular Level and on Timescale of Seconds in Living Cells

Dynamic regulation of the Rho family of small guanosine triphosphatases (GTPases) with great spatiotemporal precision is essential for various cellular functions and events1, 2. Their spatiotemporally dynamic nature has been revealed by visualization of their activity and localization in real time3. In order to gain deeper understanding of their roles in diverse cellular functions at the molecular level, the next step should be perturbation of protein activities at a precise subcellular location and timing. 
To achieve this goal, we have developed a method for light-induced, spatio-temporally controlled activation of small GTPases by combining two techniques: (1) rapamycin-induced FKBP-FRB heterodimerization and (2) a photo-caging method of rapamycin. With the use of rapamycin-mediated FKBP-FRB heterodimerization, we have developed a method for rapidly inducible activation or inactivation of small GTPases including Rac4, Cdc424, RhoA4 and Ras5, in which rapamycin induces translocation of FKBP-fused GTPases, or their activators, to the plasma membrane where FRB is anchored. For coupling with this heterodimerization system, we have also developed a photo-caging system of rapamycin analogs. A photo-caged compound is a small molecule whose activity is suppressed with a photocleavable protecting group known as a caging group. To suppress heterodimerization activity completely, we designed a caged rapamycin that is tethered to a macromolecule such that the resulting large complex cannot cross the plasma membrane, leading to virtually no background activity as a chemical dimerizer inside cells6. Figure 1 illustrates a scheme of our system. With the combination of these two systems, we locally recruited a Rac activator to the plasma membrane on a timescale of seconds and achieved light-induced Rac activation at the subcellular level6.

A method for spatio-temporal control of small GTPase activity by light is described. This method is based on rapamycin-induced FKBP-FRB heterodimerization and photo-caging systems. Optimization of light-irradiation enables the spatio-temporally controlled activation of small GTPases at the subcellular level.

Dynamic regulation of the Rho family of small guanosine triphosphatases (GTPases) with great spatiotemporal precision is essential for various cellular ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Models and Methods to Evaluate Transport of Drug Delivery Systems Across Cellular Barriers

Sub-micrometer carriers (nanocarriers; NCs) enhance efficacy of drugs by improving solubility, stability, circulation time, targeting, and release. Additionally, traversing cellular barriers in the body is crucial for both oral delivery of therapeutic NCs into the circulation and transport from the blood into tissues, where intervention is needed. NC transport across cellular barriers is achieved by: (i) the paracellular route, via transient disruption of the junctions that interlock adjacent cells, or (ii) the transcellular route, where materials are internalized by endocytosis, transported across the cell body, and secreted at the opposite cell surface (transyctosis). Delivery across cellular barriers can be facilitated by coupling therapeutics or their carriers with targeting agents that bind specifically to cell-surface markers involved in transport. Here, we provide methods to measure the extent and mechanism of NC transport across a model cell barrier, which consists of a monolayer of gastrointestinal (GI) epithelial cells grown on a porous membrane located in a transwell insert. Formation of a permeability barrier is confirmed by measuring transepithelial electrical resistance (TEER), transepithelial transport of a control substance, and immunostaining of tight junctions. As an example, ~200&#160;nm polymer NCs are used, which carry a therapeutic cargo and are coated with an antibody that targets a cell-surface determinant. The antibody or therapeutic cargo is labeled with 125I for radioisotope tracing and labeled NCs are added to the upper chamber over the cell monolayer for varying periods of time. NCs associated to the cells and/or transported to the underlying chamber can be detected. Measurement of free 125I allows subtraction of the degraded fraction. The paracellular route is assessed by determining potential changes caused by NC transport to the barrier parameters described above. Transcellular transport is determined by addressing the effect of modulating endocytosis and transcytosis pathways.

Many therapeutic applications require safe and efficient transport of drug carriers and their cargoes across cellular barriers in the body. This article describes an adaptation of established methods to evaluate the rate and mechanism of transport of drug nanocarriers (NCs) across cellular barriers, such as the gastrointestinal (GI) epithelium.

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 of such signaling can lead to many disorders. To understand cell-to-cell signaling, it is essential to study the spatial and temporal nature of the secreted molecules from the cell without disturbing the local environment. Various assays have been developed to study protein secretion, however, these methods are typically based on fluorescent probes which disrupt the relevant signaling pathways. To overcome this limitation, a label-free technique is required.
In this paper, we describe the fabrication and application of a label-free localized surface plasmon resonance imaging (LSPRi) technology capable of detecting protein secretions from a single cell. The plasmonic nanostructures are lithographically patterned onto a standard glass coverslip and can be excited using visible light on commercially available light microscopes. Only a small fraction of the coverslip is covered by the nanostructures and hence this technique is well suited for combining common techniques such as fluorescence and bright-field imaging.
A multidisciplinary approach is used in this protocol which incorporates sensor nanofabrication and subsequent biofunctionalization, binding kinetics characterization of ligand and analyte, the integration of the chip and live cells, and the analysis of the measured signal. As a whole, this technology enables a general label-free approach towards mapping cellular secretions and correlating them with the responses of nearby cells.

Inter-cellular communication is critical for controlling various physiological activities within and outside the cell. This paper describes a protocol for measuring the spatio-temporal nature of single cell secretions. To achieve this, a multidisciplinary approach is used which integrates label-free nanoplasmonic sensing with live cell imaging.

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

Recent fiber-bundle microendoscopy techniques enable non-invasive analysis of in vivo tissue using either imaging techniques or a combination of spectroscopy techniques. Combining imaging and spectroscopy techniques into a single optical probe may provide a more complete analysis of tissue health. In this article, two dissimilar modalities are combined, high-resolution fluorescence microendoscopy imaging and diffuse reflectance spectroscopy, into a single optical probe. High-resolution fluorescence microendoscopy imaging is a technique used to visualize apical tissue micro-architecture and, although mostly a qualitative technique, has demonstrated effective real-time differentiation between neoplastic and non-neoplastic tissue. Diffuse reflectance spectroscopy is a technique which can extract tissue physiological parameters including local hemoglobin concentration, melanin concentration, and oxygen saturation. This article describes the specifications required to construct the fiber-optic probe, how to build the instrumentation, and then demonstrates the technique on in vivo human skin. This work revealed that tissue micro-architecture, specifically apical skin keratinocytes, can be co-registered with its associated physiological parameters. The instrumentation and fiber-bundle probe presented here can be optimized as either a handheld or endoscopically-compatible device for use in a variety of organ systems. Additional clinical research is needed to test the viability of this technique for different epithelial disease states.

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

The assembly and use of a multimodal microendoscope is described which can co-register superficial tissue image data with tissue physiological parameters including hemoglobin concentration, melanin concentration, and oxygen saturation. This technique can be useful for evaluating tissue structure and perfusion, and can be optimized for individual needs of the investigator.

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 biodegradability, as well its ability to protect and subsequently release its payload in response to a trigger. While silk can be formulated into various material formats, silk nanoparticles are emerging as promising drug delivery systems. Therefore, this article covers the procedures for reverse engineering silk cocoons to yield a regenerated silk solution that can be used to generate stable silk nanoparticles. These nanoparticles are subsequently characterized, drug loaded and explored as a potential anticancer drug delivery system. Briefly, silk cocoons are reverse engineered first by degumming the cocoons, followed by silk dissolution and clean up, to yield an aqueous silk solution. Next, the regenerated silk solution is subjected to nanoprecipitation to yield silk nanoparticles &#8211; a simple but powerful method that generates uniform nanoparticles. The silk nanoparticles are characterized according to their size, zeta potential, morphology and stability in aqueous media, as well as their ability to entrap a chemotherapeutic payload and kill human breast cancer cells. Overall, the described methodology yields uniform silk nanoparticles that can be readily explored for a myriad of applications, including their use as a potential nanomedicine.

Nanoparticles are emerging as promising drug delivery systems for a broad range of indications. Here, we describe a simple yet powerful method to manufacture silk nanoparticles using reverse engineered Bombyx mori silk. These silk nanoparticles can be readily loaded with a therapeutic payload and subsequently explored for drug delivery applications.

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 the placenta while minimizing fetal and maternal side effects remains challenging. Targeted nanoparticle carriers provide new opportunities to treat placental disorders. We recently demonstrated that a synthetic placental chondroitin sulfate A binding peptide (plCSA-BP) could be used to guide nanoparticles to deliver drugs to the placenta. In this protocol, we describe in detail a system for assessing the efficiency of drug delivery to the placenta by plCSA-BP that employs three separate methods used in combination: in vivo imaging, high-frequency ultrasound (HFUS), and high-performance liquid chromatography (HPLC). Using in vivo imaging, plCSA-BP-guided nanoparticles were visualized in the placentas of live animals, while HFUS and HPLC demonstrated that plCSA-BP-conjugated nanoparticles efficiently and specifically delivered methotrexate to the placenta. Thus, a combination of these methods can be used as an effective tool for the targeted delivery of drugs to the placenta and development of new treatment strategies for several pregnancy complications.

We describe a system that utilizes three methods to evaluate the safety and effectiveness of placenta-targeted drug delivery: in vivo imaging to monitor nanoparticle accumulation, high-frequency ultrasound to monitor placental and fetal development, and HPLC to quantify drug delivery to tissue.

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 mimicking environment. Furthermore, the development of cell-free expression systems has demonstrated the ability to reconstitute the protein production, transcription and translation processes (DNA&#8594;RNA&#8594;protein) in a controlled manner, harnessing synthetic biology. Here we describe a protocol for preparing a cell-free expression system, including the production of a potent bacterial lysate and encapsulating this lysate inside cholesterol-rich lipid-based giant unilamellar vesicles (GUVs) (i.e., stable liposomes), to form synthetic cells. The protocol describes the methods for preparing the components of the synthetic cells including the production of active bacterial lysates, followed by a detailed step-by-step preparation of the synthetic cells based on a water-in-oil emulsion transfer method. These facilitate the production of millions of synthetic cells in a simple and affordable manner with a high versatility for producing different types of proteins. The obtained synthetic cells can be used to investigate protein/RNA production and activity in an isolated environment, in directed evolution, and also as a controlled drug delivery platform for on-demand production of therapeutic proteins inside the body.

This protocol describes the method, materials, equipment and steps for bottom-up preparation of RNA and protein producing synthetic cells. The inner aqueous compartment of the synthetic cells contained the S30 bacterial lysate encapsulated within a lipid bilayer (i.e., stable liposomes), using a water-in-oil emulsion transfer method.

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