*2.12. Di*ff*usion of Liposomes in Intact Porcine Vitreous*

Vitreal diffusion of uncoated, PEG-coated, and HA-coated liposomes in the porcine eye was investigated as reported previously [29]. In brief, fresh porcine eyes (age: six months) enucleated with a transconjunctival incision 15 min after sacrificing the animal were obtained from the slaughterhouse (HKScan Finland Oyj). After cutting out the extraocular tissues, the eyes were shortly immersed in 70% ethanol and then kept in phosphate buffered saline (PBS) buffer at 4 ◦C. The anterior segment of the eye was cut circumferentially 2 mm below the limbus, and the lens was removed. Then, 50 µL of 0.25 mg/mL fluorescently labeled liposomes was injected into the center of the exposed vitreous humor at a depth of 0.5 cm using a 30 G insulin syringe (BD, Franklin Lakes, NJ, USA). Next, a 35 mm microwell dish with a 14 mm diameter glass window (MatTek Corporation) was placed on top of the cut eyecup, while avoiding air bubble formation between the glass window and the vitreous humor. In order to prepare the eye for imaging, the dish-covered eyecup was carefully flipped over, and the edges of the eye were fixed to the dish using Loctite Precision super glue (Henkel Corp., NJ, USA). Imaging was carried out at 37 ◦C with a spinning disc confocal microscope (Marianas, Intelligent Imaging Innovation (3i) Colorado, USA) and 50 ms temporal-resolution movies were recorded using Slidebook® Software v. 6 (Intelligent Imaging Innovation). Movement of the Cy5-labeled liposomes was tracked using the single-particle tracking technique, and the trajectories were analyzed with Imaris v. 9.2 software (Bitplane AG, Zurich, Switzerland). Finally, the particle mean square displacement (MSD) was computed using the @msdanalyzer MATLAB plug-in (MathWorks Inc., Massachusetts, USA), and the vitreal diffusion coefficient of liposomes (Dv) was calculated using Equation (4).

$$\mathbf{D}\_{\rm V} = \frac{\mathbf{MSD}}{2\mathbf{d}\tau},\tag{4}$$

where MSD is derived from the slope of the MSD curve, *d* is defined as dimensionality of the movement (*d* = 2 for 2D tracks), and τ is the lag time of the calculated displacement. The results are reported as Dw/Dv, which indicates how many times more rapidly the liposomes diffuse in water compared to the more viscous vitreous gel. The theoretical diffusion in water (Dw) was calculated according to the Stokes–Einstein equation at 37 ◦C, and the radii of liposome particles were obtained from DLS measurements as described above.
