Description

There are five fluorescent control liposome products (Fluoroliposome®) for Clodrosome® (clodronate liposomes). All five fluorescent liposomes incorporate a lipophilic dye inside their membranes. They are insoluble in water; however, their fluorescence is easily detected when incorporated into membranes. DiI, DiO, DiD, DiR and DiA cover a wide range of excitation and emission wavelengths from 300s to 900s. DiI and DiO have fluorescence excitation and emission maxima separated by about 65 nm, facilitating two-color labeling. The emission spectrum of DiA is very broad, allowing it to be detected as green, orange or even red fluorescence depending on the optical filter used. DiI, DiO, DiD and DiR belong to the dialkylcarbocyanines family of compounds. The spectral properties of the dialkylcarbocyanines are largely independent of the lengths of the alkyl chains. Instead, they are determined by the heteroatoms in the terminal ring systems and the length of the connecting bridge. They have extremely high extinction coefficients, moderate fluorescence quantum yields and short excited state lifetimes in lipid environments (~1 ns). The fluorescence spectrum of each dye is shown below.

You can choose the Fluoroliposome® based on the type of the fluorescent equipment and filters that you use in your lab. Clodronate liposomes cannot be made fluorescent simply due to the potential for inaccurate and/or uninterpretable data being generated by labelled Clodrosome®. For more information, please refer to the technical note section.

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Technical Information

Five-Color Fluoroliposome® Kit

Lipid Composition	Concentration (mg/ml)	Concentration (mM)	Molar Ratio Percentage
Total	23 mg/ml	35.1 mM	100
L-alpha-Phosphatidylcholine	18.8	24.3	70
Cholesterol	4.2	10.9	30

Fluorescent Dye	Excitation/Emission (nm)	Concentration (mg/ml)	Concentration (mM)
4-(4-(Dihexadecylamino)styryl)-N-Methylpyridinium Iodide (DiA)	456/590	0.0625	0.0794
1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindodicarbocyanine, 4-Chlorobenzenesulfonate Salt (DiD)	644/665	0.0625	0.065
1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindocarbocyanine Perchlorate (DiI)	549/565	0.0625	0.065
3,3'-Dilinoleyloxacarbocyanine Perchlorate (DiO)	484/501	0.0625	0.071
1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindotricarbocyanine Iodide (DiR)	750/780	0.00625	0.00651

Buffer and Liposome Size	Specification
Buffer	Phosphate Buffered Saline
pH	7.4
Liposome Size	1.5-2 µm

Technical Notes

• The issue with fluorescent Clodrosome® has to do with the potential for inaccurate and/or uninterpretable data being generated by labelled Clodrosome®. When Clodrosome® induces macrophage apoptosis, the fluorescent lipid incorporated into the Clodrosome® is disrupted and metabolized in the phagolysosome will be dispersed among the residual apoptotic bodies which are subsequently phagocytosed by other macrophages. Therefore, fluorescent lipids may be detected in phagocytic cells which never phagocytosed Clodrosome® especially when FACS or fluoroscopy are utilized to detect fluorescent cells (FACS) or fluorescence levels in a tissue homogenate (fluoroscopy). Another potential artifact arises from fluorescent lipid remaining in the extracellular “garbage”, which has not yet been cleared by other phagocytes, generating a high background fluorescence. However, experienced confocal microscopist may be able to differentiate between the punctate fluorescence, resulting from fluorescent intact liposomes versus the more diffuse fluorescence characteristic of disrupted liposomes and some have successfully used fluorescent clodronate liposomes to visualize the cellular location of these liposomes by confocal microscopy in vivo [1]. A further complicating factor is that published data varies widely as to exactly when clodronate liposomes begin to induce apoptosis in macrophages. Mönkönnnen et al. show that macrophage death is measurable within the first hour after clodronate liposome treatment on RAW264 cells in vitro [2], while many others have reported no signs of macrophage apoptosis until several hours after treatment in vivo. The variability in the data is likely due to different liposomal formulations of clodronate as well as the vastly different experimental conditions. Therefore, as with most biological studies, especially those involving liposomes, the amount of time between treating the animal or cells with clodronate liposomes and the onset of apoptosis will need to be established in each experimental model. If the nature of the research demands that Clodrosome® be tracked rather than the control, Encapsula can provide DiI-labelled Clodrosome® upon request, and assuming that the Clodrosome® distribution can definitively be assessed prior to the onset of apoptosis, clear and valid data on the biodistribution of fluorescent Clodrosome® should be obtainable. Still, for most purposes, Fluoroliposome® (fluorescent control liposomes) will provide the required data with far fewer potential artifacts.
• When monitoring monocyte uptake in vivo in normal animals, the circulating monocytes may “disappear” or show reduced counts within the first 2 h post-injection due to margination of the monocytes post-liposome phagocytosis. These cells will re-enter the circulation within a few hours. Sunderkötter et al. demonstrate this phenomenon and discuss the behavior in detail. Also consider that circulating monocytes have a lifetime of about 24 h so labeled monocytes will be continually leaving the circulation, even in normal animals, due to aging of the monocytes [3].
• Liposomes may settle when left undisturbed for more than a few hours. Immediately prior to use, in order to ensure a homogeneous liposome suspension, slowly invert the vial several times until the suspension appears homogeneous by visual inspection. Vigorous or erratic shaking will not damage the liposomes but may induce foaming and bubble formation making it more difficult to accurately measure the desired dosage.
• If the personnel performing intravenous injections are not experienced in or familiar with, precautions for injecting larger volumes (~10% animal weight in ml), viscous liquids or particulate suspensions, consider having extra animals available in case serious injection-related adverse events occur. Dose control animals first to become familiar with large volume injections.
• When dosing intravenously, use standard precautions for dosing larger volumes to animals including the following: a) Warm product to room temperature prior to dosing. b) Ensure that all air bubbles are removed from the syringe prior to dosing; intravenous injection of air bubbles may result in air emboli which can kill or seriously injure animals. c) Inject product at a slow, steady rate of no more than 1 ml/min; decrease infusion rate if animals display any atypical reactions such as unusual agitation.
• Infusion-related adverse reactions usually involve the animal gasping for air or other seizure-like movements. Animals often recover with no apparent permanent injury, but any potential effects on experimental results must be assessed by the researcher.
• Liposomes should be kept at 4°C and NEVER be frozen.

Dosage

Appearance

Fluoroliposome®-DiD, Fluoroliposome®-DiI and Fluoroliposome®-DiR are blue, pink and dark blue liquid suspensions, respectively. Fluoroliposome®-DiA and Fluoroliposome®-DiO are yellow liquid suspensions. These Fluoroliposome® products are made of large micron size multilamellar liposomes. Due to their large size, some liposomes might settle to the bottom of the vial. If left sitting idle in the refrigerator, they will phase separate and form pellets in the bottom of the vial, leaving a clear solution on top. Therefore, the vial should be shaken to form a homogeneous solution prior to use.

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Ordering/Shipping Information

• All liposome based formulations are shipped on blue ice at 4°C in insulated packages using overnight shipping or international express shipping.
• Liposomes should NEVER be frozen. Ice crystals that form in the lipid membrane can rupture the membrane, change the size of the liposomes and cause the encapsulated drug to leak out. Liposomes in liquid form should always be kept in the refrigerator.
• Clients who order from outside of the United States of America are responsible for their government import taxes and customs paperwork. Encapsula NanoSciences is NOT responsible for importation fees to countries outside of the United States of America.
• We strongly encourage the clients in Japan, Korea, Taiwan and China to order via a distributor. Tough customs clearance regulations in these countries will cause delay in custom clearance of these perishable formulations if ordered directly through us. Distributors can easily clear the packages from customs. To see the list of the distributors click here.
• Clients ordering from universities and research institutes in Australia should keep in mind that the liposome formulations are made from synthetic material and the formulations do not require a “permit to import quarantine material”. Liposomes are NOT biological products.
• If you would like your institute’s FedEx or DHL account to be charged for shipping, then please provide the account number at the time of ordering.
• Encapsula NanoSciences has no control over delays due to inclement weather or customs clearance delays. You will receive a FedEx or DHL tracking number once your order is confirmed. Contact FedEx or DHL in advance and make sure that the paperwork for customs is done on time. All subsequent shipping inquiries should be directed to Federal Express or DHL.

Storage and Shelf Life

Storage

Fluoroliposome® products should always be stored at in the dark at 4°C, except when brought to room temperature for brief periods prior to animal dosing. DO NOT FREEZE. ENS is not responsible for results generated by frozen product.

Shelf Life

Fluoroliposome® products are made on daily basis. The batch that is shipped is manufactured on the same day. It is advised to use the products within 60 days of the manufacturing date.

References and background reading

1. Polfliet MM, Goede PH, van Kesteren-Hendrikx EM, van Rooijen N, Dijkstra CD, van den Berg TK. A method for the selective depletion of perivascular and meningeal macrophages in the central nervous system. J. Neuroimmunol. 2001 Jun 1;116(2):188–95.

2. Mönkkönen J, Liukkonen J, Taskinen M, Heath TD, Urtti A. Studies on liposome formulations for intra-articular delivery of clodronate. Journal of Controlled Release. 1995 Aug;35(2–3):145–54.

3. Sunderkötter C, Nikolic T, Dillon MJ, van Rooijen N, Stehling M, Drevets DA, Leenen P. Subpopulations of Mouse Blood Monocytes Differ in Maturation Stage and Inflammatory Response. J Immunol. 2004 Apr 1;172(7):4410–7.

4. Nagai H, Kuwahira I, Schwenke DO, Tsuchimochi H, Nara A, Ogura S, Sonobe T, Inagaki T, Fujii Y, Yamaguchi R, Wingenfeld L. Pulmonary macrophages attenuate hypoxic pulmonary vasoconstriction via β3AR/iNOS pathway in rats exposed to chronic intermittent hypoxia. PLoS One. 2015 Jul 1;10(7):e0131923.

5. Zhu Y, Soderblom C, Krishnan V, Ashbaugh J, Bethea JR, Lee JK. Hematogenous macrophage depletion reduces the fibrotic scar and increases axonal growth after spinal cord injury. Neurobiology of disease. 2015 Feb 28;74:114-25.

6. Yun MH, Davaapil H, Brockes JP. Recurrent turnover of senescent cells during regeneration of a complex structure. Elife. 2015;4:e05505.

7. Arwert EN, Harney AS, Entenberg D, Wang Y, Sahai E, Pollard JW, Condeelis JS. A Unidirectional Transition from Migratory to Perivascular Macrophage Is Required for Tumor Cell Intravasation. Cell reports. 2018 May 1;23(5):1239-48.