This work was supported by the Deutsche Forschungsgemeinschaft grants HI-698/10-1 and RU-1652/1-1. We thank Doreen May for excellent technical assistance and the company DYOMICS GmbH, Jena for their kind support.

NOTE: All procedures are approved by the regional animal committee and in accordance with international guidelines on the ethical use of animals.
1. Preparation of Materials and Instruments
<ol>
	<li>Preparation of spontaneously formed vesicle dispersion (SFV)
	<ol>
		<li>Dissolve and prepare stock solutions of the following phospholipids: 214 mg/ml egg phosphatidylcholine (EPC), 134 mg/ml cholesterol, 122 mg/ml 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(pol...

Since liposomes can also serve as delivery systems for fluorescent dyes, they enable imaging of target diseases. The encapsulation of high concentrations of fluorescent dyes such as the NIRF dye, DY676-COOH used here, leads to a high level of fluorescence quenching of the entrapped dye. Fluorescence quenching, a phenomenon seen with many fluorophores at high concentration can be exploited in several in vivo imaging applications, where a high sensitivity and reliable detection of the target area is demanded. The ...

Liposomes have been intensively investigated and serve as one of the most biocompatible biomedical drug delivery systems for clinical applications1,2. They are mainly composed of phospholipids and cholesterol, both of which are biocompatible compounds mimicking parts of natural cell membranes. Whereas hydrophilic substances can be entrapped in the aqueous interior, lipophilic agents can be incorporated within the liposomal phospholipid bilayer3. Encapsulation of substances within the aqueous interior of liposomes grants protection against degradation in vivo and also prevents the host system from toxic effects of cytotoxic drugs used for the therapy of diseases, for example chemotherapeutics aimed at destroying tumor cells. The modification of the liposomal surface with polymers like polyethylenglycol (PEGylation) further extends the liposomal blood circulation time in vivo due to sterical stabilization4. Moreover, liposomes can sequester high concentrations of several substances such as proteins5,6, hydrophilic substances7,8 and enzymes9. They therefore serve as reliable clinical therapeutic and diagnostic tools which merit their approval for delivery of cytotoxic drugs such as doxorubicin for cancer therapy4. Due to their flexibility, liposomes can also be loaded with fluorochromes for diagnostic and image-guided surgical purposes.
Fluorescence imaging provides a cost-effective and non-invasive in vivo diagnostic tool which however, demands some basic requirements. It could be demonstrated that fluorochromes which suit best for in vivo imaging have characteristic absorption and emission maxima in the range where light dispersion and scattering as well as tissue autofluorescence originating from water and hemoglobin is low. Thus, such probes have their abs/em maxima between 650 and 900 nm10. Besides this, the stability of fluorochromes both in vitro and in vivo is critical, as opsonization and rapid clearance can greatly limit their application for in vivo imaging11. Other effects such as poor stability and low sensitivity or cytotoxic effects on target organs as seen with indocyanine green (ICG)12-16, are unwanted and must be taken into consideration when designing probes for in vivo imaging. These observations have led to the active development of several preclinical NIR fluorochromes, nanoparticles as well as new techniques for the in vivo imaging of inflammatory processes, cancer and for image-guided surgery17-20. Despite the stability of most preclinical NIRF (near-infrared fluorescence) dyes in vitro, their rapid perfusion and clearance through the liver and kidney impede their use in the in vivo optical imaging of diseases and inflammatory processes.
We therefore present a protocol for the encapsulation of fluorochromes such as the well characterized near-infrared fluorescent dye DY-676-COOH, known for its tendency to self-quench at relatively high concentrations21 in liposomes. At high concentrations H-dimer formation and/or pi-stacking interactions between fluorophore molecules located within each other&#8217;s F&#246;rster radius result in F&#246;rster resonance energy transfer (FRET) between the fluorochrome molecules. At low concentration the space between the fluorophore molecules increases, thereby preventing pi-stacking interaction and H-dimer formation and resulting in high fluorescence emission. The switch between high and low concentration and the accompanying fluorescence quenching and activation is a promising strategy that can be exploited for optical imaging22. In this respect, encapsulation of high concentrations of the NIRF dye DY-676-COOH in the aqueous interior of liposomes is more favorable for in vivo imaging than the free dye. The challenge of the method lies first of all in the correct encapsulation and secondly, in the validation of the benefits resulting from encapsulating high concentrations of the dye. Comparing the imaging properties of quenched liposomes with that of the free dye and also with a non-quenched liposome formulation with low concentrations of the dye is indispensable. We show by a simple, but highly effective film hydration and extrusion protocol combined with alternate freeze and thaw cycles that encapsulation of quenching concentrations of DY-676-COOH in liposomes is feasible. Other methods used to prepare liposomes such as the reversed phase evaporation method23 as well as the ethanol injection method24 enable liposome preparation with high encapsulation efficiencies for many hydrophilic substances. However, the nature of the substance to be encapsulated can influence the encapsulation efficiency. In effect, the film hydration and extrusion protocol presented here revealed the highest efficiency for encapsulation of DY-676-COOH. To illustrate the benefits of liposomal encapsulation of DY-676-COOH, a zymosan-induced edema model, which permits the study of inflammatory processes within a few hours, was used. Here, it is demonstrated that liposomes with high concentrations of the encapsulated DY-676-COOH are more suitable for whole body in vivo optical imaging of inflammatory processes than the free dye or the non-quenched liposomal formulation with low dye concentrations. Thus the underlying protocol provides a simple and fast method to produce quenched fluorescent liposomes and the validation of their activation and imaging potential both in vitro and in vivo.

<table border="0" cellpadding="0" cellspacing="0" width="872">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">Name of Material/ Equipment</td>
			<td style="width:179px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:267px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:309px;">Materials and equipments for preparation of liposomes</td>
			<td style="width:179px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">egg phospahtidylcholine</td>
			<td>Avanti Polar Lipids</td>
			<td>840051P</td>
			<td>Dissolve in Chloroform and store in glass vials (214 mg/ml)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">cholesterol</td>
			<td>Sigma</td>
			<td>C8667</td>
			<td>Dissolve in Chloroform and store in glass vials (134 mg/ml)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt)</td>
			<td>Avanti Polar Lipids</td>
			<td>880120P</td>
			<td>Dissolve in Chloroform and store in glass vials (122 mg/ml)</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:309px;">1,2-dioleoyl-snglycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (ammonium salt)</td>
			<td>Avanti Polar Lipids</td>
			<td>810145P</td>
			<td>Dissolve in Chloroform and store in glass vials (2mg/ml)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">Sartorius MC1 (d = 0.01 mg)</td>
			<td>Sartorius AG</td>
			<td>Research RC 210 P</td>
			<td>used for weighing the phospholipids</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:309px;">Rotavapor</td>
			<td>B&#252;chi Labortechnik AG</td>
			<td>R-114</td>
			<td>used for hydration of phospholipid film</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:309px;">Waterbath</td>
			<td>B&#252;chi Labortechnik AG</td>
			<td>R-481</td>
			<td>used for hydration of phospholipid film</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:309px;">Vacuum Controller</td>
			<td>B&#252;chi Labortechnik AG</td>
			<td>B-720</td>
			<td>used for hydration of phospholipid film</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:309px;">Vacobox</td>
			<td>B&#252;chi Labortechnik AG</td>
			<td>B-177</td>
			<td>used for hydration of phospholipid film</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">Circulation Chiller</td>
			<td style="width:179px;">LAUDA DR. R. WOBSER 
			GMBH &#38; CO. KG</td>
			<td>WKL 230</td>
			<td>used for hydration of phospholipid film</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DY-676-COOH</td>
			<td>Dyomics GmbH</td>
			<td>676-00</td>
			<td>Dissolve in 10 mM Tris and store stock at -20&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris-(Hydroxymethyl)-aminomethan</td>
			<td>Applichem</td>
			<td>A1086</td>
			<td>buffer 10 mM, pH 7.4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trichlormethan</td>
			<td>Carl Roth GmbH + Co. KG</td>
			<td>Y015.2</td>
			<td>used for liposome preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sonicator</td>
			<td>Merck Eurolab GmbH</td>
			<td>USR 170 H</td>
			<td>used for liposome preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vortex Genie 2 (Pop-off Cup, No. 146-3011-00)</td>
			<td>Scientific Industries Inc.</td>
			<td>SI-0256</td>
			<td>used for liposome preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sephadex G25 medium&#160;</td>
			<td>GE Healthcare Europe GmbH</td>
			<td>17-0033-01</td>
			<td>used for liposome purification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X100</td>
			<td>Ferak Berlin GmbH</td>
			<td>505002</td>
			<td>used to destruct liposomes&#160; for dye quantification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LiposoFast-Basic</td>
			<td>Avestin Inc.</td>
			<td style="width:119px;"></td>
			<td>used for the extrusion of liposomes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polycarbonate filter membrane, 100 nm (Whatman Nucleopore Trans Etch Membrane, NUCLEPR PC 19 MM, 0.1 U)</td>
			<td>VWR</td>
			<td style="width:119px;"></td>
			<td>used for the extrusion of liposomes via LiposoFast-Basic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluostar Optima</td>
			<td>BMG Labtech</td>
			<td style="width:119px;"></td>
			<td>used for dye quantification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Zetasizer Nano ZS</td>
			<td>Malvern</td>
			<td style="width:119px;"></td>
			<td>used for the determination of liposome size and zetapotential</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">Ultracentrifuge&#160;</td>
			<td style="width:179px;">Beckmann Coulter GmbH</td>
			<td style="width:119px;">XL 80</td>
			<td>used for concentration of the samples</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">Rotor</td>
			<td style="width:179px;">Beckmann Coulter GmbH</td>
			<td style="width:119px;">SW 55 TI</td>
			<td>used for concentration of the samples</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;"></td>
			<td style="width:179px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:309px;">Materials and equipments for the evaluation of liposome and optical imaging&#160;</td>
			<td style="width:179px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;"></td>
			<td style="width:179px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">Zymosan-A from Saccharomyces cereviciae</td>
			<td style="width:179px;">Sigma</td>
			<td style="width:119px;">Z4250-250MG</td>
			<td>used for induction of inflammation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">Isotonic Saline (0.9)</td>
			<td style="width:179px;">Fresenius GmbH</td>
			<td style="width:119px;">PZN-2159621</td>
			<td>used for the dilution of Zymosan-A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">Isoflurane vaporizer</td>
			<td>Ohmeda Isotec 4</td>
			<td style="width:119px;"></td>
			<td>used for anesthesizing animals</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">Isoflurane</td>
			<td>Actavis GmbH&#160;</td>
			<td style="width:119px;">PZN-7253744</td>
			<td>anesthesia</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thermo Mat Pro 20 W</td>
			<td style="width:179px;">Lucky Reptile</td>
			<td style="width:119px;">61202-HTP-20</td>
			<td>used to keep animals warm during anesthesia</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">Omnican-F (1 ml injection)&#160;</td>
			<td style="width:179px;">Braun</td>
			<td style="width:119px;">PZN-3115465</td>
			<td>used for subcutaneous and intravenous application of probes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">Panthenol eye cream</td>
			<td style="width:179px;">Jenapharm</td>
			<td style="width:119px;">PZN-3524531</td>
			<td>used to prevent dryness of the eyes of animals during anesthesia</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:309px;">Hanks buffered saline solution</td>
			<td style="width:179px;">PAA Laboratories /Biochrom AG</td>
			<td style="width:119px;">L2045</td>
			<td>w/o Mg2+, Ca2+ and phenol red. For dilution of probes and for washing of cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">8-Well chamber slides</td>
			<td style="width:179px;">BD Biosciences</td>
			<td style="width:119px;">354108</td>
			<td>used for cell culture followed by microscopy&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">Cell culture flasks</td>
			<td style="width:179px;">Greiner BioOne</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell culture media</td>
			<td colspan="2">Gibco (life technologies GmbH)</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal calf serum&#160;</td>
			<td>Invitrogen</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Poly-L-Lysine solution (0,01% - 50&#160;ml)</td>
			<td>Sigma</td>
			<td style="width:119px;">P4832</td>
			<td>used to coat cell culture chamber slides</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mountant Permafluor</td>
			<td>ThermoScientific&#160;</td>
			<td>S21022-3</td>
			<td>Mounting solution for microscopy</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">Hoechst-33258</td>
			<td style="width:179px;">AppliChem</td>
			<td style="width:119px;"></td>
			<td>DNA stain for microscopy</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;"></td>
			<td style="width:179px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">Hera-Safe</td>
			<td>Heraeus Instruments</td>
			<td style="width:119px;"></td>
			<td>sterile work bench used for cell culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HERA cell</td>
			<td>Heraeus Instruments</td>
			<td style="width:119px;"></td>
			<td>Incubator used for cell culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">LSM510-Meta</td>
			<td style="width:179px;">Zeiss</td>
			<td style="width:119px;"></td>
			<td>used for confocal microscopy</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:309px;">Maestro-TM in vivo fluorescence imaging system</td>
			<td style="width:179px;">CRi, Woburn</td>
			<td style="width:119px;"></td>
			<td>used for whole body fluorescence imaging of small animals</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">Spectrophotometer (Ultrospec 4300 pro UV)</td>
			<td style="width:179px;">GE Healthcare</td>
			<td style="width:119px;"></td>
			<td>used for measurement of absorption</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">Spectrofluorometer (Jasco FP-6200)</td>
			<td style="width:179px;">Jasco</td>
			<td style="width:119px;"></td>
			<td>used for measurement of fluorescence emission</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;"></td>
			<td style="width:179px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">Animals</td>
			<td style="width:179px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NMRI mice (8-12 weeks old, male)</td>
			<td style="width:179px;">Elevage Janvier, France</td>
			<td style="width:119px;"></td>
			<td>used for inflammation trials</td>
		</tr>
	</tbody>
</table>

fluorescence-quenching of a liposomal-encapsulated near-infrared fluorophore as a tool for in vivo optical imaging

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous number of imaging techniques currently available and the progress in instrumentation, there is still a need for highly sensitive probes that are suitable for in vivo imaging. One typical problem of available preclinical fluorescent probes is their rapid clearance in vivo, which reduces their imaging sensitivity. To circumvent rapid clearance, increase number of dye molecules at the target site, and thereby reduce background autofluorescence, encapsulation of the near-infrared fluorescent dye, DY-676-COOH in liposomes and verification of its potential for in vivo imaging of inflammation was done. DY-676 is known for its ability to self-quench at high concentrations. We first determined the concentration suitable for self-quenching, and then encapsulated this quenching concentration into the aqueous interior of PEGylated liposomes. To substantiate the quenching and activation potential of the liposomes we use a harsh freezing method which leads to damage of liposomal membranes without affecting the encapsulated dye. The liposomes characterized by a high level of fluorescence quenching were termed Lip-Q. We show by experiments with different cell lines that uptake of Lip-Q is predominantly by phagocytosis which in turn enabled the characterization of its potential as a tool for in vivo imaging of inflammation in mice models. Furthermore, we use a zymosan-induced edema model in mice to substantiate the potential of Lip-Q in optical imaging of inflammation in vivo. Considering possible uptake due to inflammation-induced enhanced permeability and retention (EPR) effect, an always-on liposome formulation with low, non-quenched concentration of DY-676-COOH (termed Lip-dQ) and the free DY-676-COOH were compared with Lip-Q in animal trials.

The encapsulation of high concentrations of fluorescent dyes such as the NIRF dye DY676-COOH used here in the aqueous interior of liposomes leads to a high level of fluorescence quenching. Fluorescence quenching, a phenomenon seen with many fluorophores at high concentration, can be exploited in several in vivo imaging applications where a high sensitivity and reliable detection of the target area are demanded. The use of liposomes also provides protection of the dye which is indispensable for in vivo a...

Watch this Scientific Journal Video about Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging at JoVE.com

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

The use of fluorophores for in vivo imaging can be greatly limited by opsonization, rapid clearance, low detection sensitivity and cytotoxic effects on the host. Encapsulation of fluorophores in liposomes by film hydration and extrusion leads to fluorescence quenching and protection which enables in vivo imaging with high detection sensitivity.

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous ...

fluorescence-quenching-liposomal-encapsulated-near-infrared

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JoVE Journal

Bioengineering

16.1K ビュー.  Jena University Hospital. The use of fluorophores for in vivo imaging can be greatly limited by opsonization, rapid clearance, low detection sensitivity and cytotoxic effects on the host. Encapsulation of fluorophores in liposomes by film hydration and extrusion leads to fluorescence quenching and protection which enables in vivo imaging with high detection sensitivity. 