Cyanine5.5 amine (Cy 5.5 amine), a Cy5.5 analog, is a near-infrared (NIR) fluorescent dye (Ex=648 nm, Em=710 nm). Cyanine5.5 amine may be utilized to prepare Cy5.5-labeled nanoparticles that can be tracked and imaged with confocal microscopy with low fluorescence background.

Physicochemical Properties

Molecular Formula	C46H58CL2N4O
Molecular Weight	753.8849
Exact Mass	752.398
CAS #	2097714-45-7
Related CAS #	2097714-44-6 (without anion);2097714-45-7 (chloride);
PubChem CID	165412490
Appearance	Purple to purplish red solid powder
Hydrogen Bond Donor Count	3
Hydrogen Bond Acceptor Count	4
Rotatable Bond Count	15
Heavy Atom Count	53
Complexity	1290
Defined Atom Stereocenter Count	0
SMILES	[Cl-].[Cl-].O=C(CCCCCN1C2C=CC3=CC=CC=C3C=2C(C)(C)/C/1=C\C=C\C=C\C1C(C)(C)C2C3=CC=CC=C3C=CC=2[N+]=1C)NCCCCCC[NH3+]
InChi Key	KVJPEAWDFIBHDL-UHFFFAOYSA-N
InChi Code	InChI=1S/C46H56N4O.2ClH/c1-45(2)40(49(5)38-29-27-34-20-13-15-22-36(34)43(38)45)24-10-8-11-25-41-46(3,4)44-37-23-16-14-21-35(37)28-30-39(44)50(41)33-19-9-12-26-42(51)48-32-18-7-6-17-31-47;;/h8,10-11,13-16,20-25,27-30H,6-7,9,12,17-19,26,31-33,47H2,1-5H3;2*1H
Chemical Name	N-(6-aminohexyl)-6-[(2E)-1,1-dimethyl-2-[(2E,4E)-5-(1,1,3-trimethylbenzo[e]indol-3-ium-2-yl)penta-2,4-dienylidene]benzo[e]indol-3-yl]hexanamide;chloride;hydrochloride
HS Tariff Code	2934.99.9001
Storage	Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Biological Activity

ln Vivo

Cy5.5-labeled nanoparticles (Cy5.5-PLGA) in retinal blood vessels: real-time monitoring. Advice (This is our suggested protocol, which should be adjusted to meet your unique requirements as it only offers guidance) [2]. 1. Give freshly made Cy5.5-PLGA suspension intravenously (0.5 mL); resuspend in 0.5 ml of 1% Poloxamer 188; vortex gently and let sit at room temperature for 30 minutes before using. 2. Rats are anesthetized, the iris is relaxed with 5% neosynephrine-POS, and Vidisic eye gel is applied to prevent dry eyes and to act as a soaking medium for contact lenses. 3. With the rat's eyes at working distance below the objective lens, place it under a confocal scanning microscope and place the cannula into the vein in its tail. 4. Take pictures and record retinal fluorescence at various intervals (0, 1, 3, 5, 15, 30, 60, 90 minutes). NOTE: Throughout every step of the in vivo imaging process, the rats stay on the heating plate. 5. The rat should be put to death with an overdose of the aforementioned anesthetic after in vivo real-time imaging. The eyeball should then be removed and placed in a cooled HEPES buffer solution (135 mM NaCl, 5 mM NaOH, 2.5 mM KCl, 7 mM MgCl2, 10 mM HEPES, 10 mM glucose; pH7.4). 6. The entire retina should be carefully separated, the anterior segment and vitreous body removed, and it should be laid flat on a modified culture plate. 7. To stain the nuclei, incubate the entire mount in 0.1 mg/mL Hoechst 33342 in HEPES solution for 20 minutes. 8. After 20 minutes of fixing flat-mounted retinas with 4% paraformaldehyde solution, wash with HEPES solution. 9. As soon as the retinal plain slide is ready, use the microscope to take a picture.

References

[1]. Intracellular Uptake Mechanism of Bioorthogonally Conjugated Nanoparticles on Metabolically Engineered Mesenchymal Stem Cells. Bioconjug Chem. 2021 Jan 20;32(1):199-214. [2]. Release kinetics of fluorescent dyes from PLGA nanoparticles in retinal blood vessels: In vivo monitoring and ex vivo localization. Eur J Pharm Biopharm. 2020 May;150:131-142.

Solubility Data

Solubility (In Vitro)

DMSO : ~125 mg/mL (~165.81 mM)

Solubility (In Vivo)

Solubility in Formulation 1: 2.08 mg/mL (2.76 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: 2.08 mg/mL (2.76 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	1.3265 mL	6.6324 mL	13.2647 mL
	5 mM	0.2653 mL	1.3265 mL	2.6529 mL
	10 mM	0.1326 mL	0.6632 mL	1.3265 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.