Cyanine5.5 amine

Protocol for real-time monitoring of Cyanine5.5 amine-labeled nanoparticles (Cy5.5-PLGA) in retinal blood vessels^[1]:

1. Preparation of nanoparticle suspension

1) Dissolve Cyanine5.5 amine (4mg, 5.3μmol) in 8mL DCM, add PLGA solution (2400mg in 20mL DCM), add N,N-diisopropylethylamine (0.8 mg, 6.2μmol) and EDC (7.6mg, 49μmol), stir at room temperature in darkness for 48h.

2) Wash the organic phase three times with deionised water, dry over anhydrous Na₂SO₄, dissolve in ethyl acetate, precipitate with hexane, filter and dry under vacuum to yield the conjugate in the form of light-blue powder.

3) Mix Cy5.5-labelled PLGA with unlabelled PLGA at a 1:1 mass ratio and dissolve together in DCM to give the polymer solution.

4) Prepare a nanoparticle suspension from the polymer solution by a high pressure o/w emulsion solvent evaporation technique.

5) Remove non-encapsulated free dye by centrifugation or gel permeation chromatography, add 1% mannitol as cryoprotector and freeze-dry.

6) Resuspend the freshly prepared Cy5.5-PLGA nanoparticle powder in 0.5mL 1% poloxamer 188, vortex gently and leave at room temperature for 30min to allow full dispersion.

2. In vivo injection and real-time imaging

1) Anaesthetise rat, apply 1-2 drops of Neosynephrine-POS 5% to both eyes for relax the iris, and Vidisic® eye gel was applied to protect the eye from drying out and used as immersion medium for the contact lens as well.

2) Fix the rat under the confocal scanning microscope, insert a cannula into the tail vein and connect the syringe.

3) Inject 0.5mL nanoparticle suspension intravenously (slow push over 30-60s).

4) Observe retinal fluorescence and capture images at 0, 1, 3, 5, 15, 30, 60 and 90min.

Note: keep the rat on a heating plate during all the in vivo imaging process.

3. In vitro retinal imaging

1) After real-time imaging, euthanise the rat with an overdose of the above anaesthetic, enucleate the eye and place it into cooled HEPES buffer (135mM NaCl, 5mM NaOH, 2.5mM KCl, 7mM MgCl₂, 10mM HEPES, 10mM glucose, pH=7.4).

2) Remove the anterior segment and vitreous body, carefully isolate the whole retina and flatten it on a modified culture plate.

3) Incubate the whole mount with 0.1mg/mL Hoechst 33342 in HEPES solution for 20min.

4) Fix the flat-mounted retina with 4% paraformaldehyde for 20min, then rinse with HEPES solution.

5) Image the flat-mounted retina immediately under the microscope.

This protocol only provides a guide, please modify it to meet your specific needs.

References:
[1] Zhang E, Zhukova V, Semyonkin A, et al. Release kinetics of fluorescent dyes from PLGA nanoparticles in retinal blood vessels: In vivo monitoring and ex vivo localization. Eur J Pharm Biopharm. 2020;150:131-142.