Ultrasound-enhanced penetration through sclera depends on frequency of sonication and size of macromolecules.

We previously employed ultrasound as a needleless approach to deliver macromolecules via the transscleral route to the back of the eye in live animals (Suen et al., 2013). Here, we investigated the nature of the ultrasound-enhanced transport through sclera, the outermost barrier in the transscleral route. Thus, the possible role of cavitation from ultrasound was explored; its effect during and after sonication on scleral penetration was measured; and the dependence on the size of macromolecules was determined. We applied ultrasound frequency from 40kHz to 3MHz at ISATA (spatial-average-temporal-average intensity) of 0.05W/cm2 to fresh rabbit sclera ex vivo. Fluorescent dextran of size 20kDa to 150kDa was used as macromolecular probes. We measured the distance of penetration of the probes through the sclera over 30s during sonication and over 15min after sonication from cryosectioned tissue images. Deeper penetration in the sclera was observed with decreasing frequency. The presence of stable cavitation was further verified by passive acoustic detection. The effect during sonication increased penetration distance up to 20 fold and was limited to macromolecular probes ≤70kDa. The effect post sonication increased penetration distance up to 3 fold and attributed to the improved intrasscleral transport of macromolecules ≥70kDa. Post-sonication enhancement diminished gradually in 3h. As the extent of cavitation increased with decreasing frequency, the trend observed supports the contribution of (stable) cavitation to enhancing transport through sclera. Effect during sonication was attributed to flow associated with acoustic microstreaming. Effect post sonication was attributed to the temporary increase in scleral permeability. Flow-associated effect was more pronounced but only applied to smaller macromolecules.

Examination of Effects of Low-Frequency Ultrasound on Scleral Permeability and Collagen Network.

Delivery of therapeutics to the intraocular space or to targeted tissues in the posterior segment is challenging because of the structural and dynamic barriers surrounding the eye. Previously, we reported the feasibility of using ultrasound (US) irradiation to deliver macromolecules to the posterior segment of the eye via the transscleral route, which consists of sclera as the outermost anatomic barrier. In this study, we found that although ultrasound increases scleral permeability for macromolecules, the scleral collagen arrangement remains undisturbed. In an ex vivo experiment, protein permeation across the sclera was significantly enhanced by ultrasound in the stable cavitation regime. The scleral collagen network was further examined by second harmonic generation imaging. Quantitative image analysis techniques were adopted to examine the density, anisotropy and interlacing pattern of collagen fibers before and after ultrasound irradiation. Repeated ultrasound applications did not induce significant changes in the arrangement of collagen fibrils at 40 kHz with a spatial average temporal average intensity (ISATA) <1.8 W/cm2. These parameters correspond to a mechanical index (MI) below 0.8 in our setting. These data suggested that enhanced permeation of macromolecules across the sclera was achieved without disturbing the collagen network of the sclera. This evidence supports that low-frequency, low-intensity ultrasound is a tolerable approach to transscleral drug delivery.

Ultrasound-mediated transscleral delivery of macromolecules to the posterior segment of rabbit eye in vivo.

PURPOSE: This study aims to determine the in vivo effectiveness of low-frequency ultrasound in mediating the transport of macromolecules to the posterior segment of the eye via transscleral route. It investigates if damage is caused by ultrasound at the tested operation parameters on the posterior ocular tissues and visual function. METHODS: Ultrasound (I(SATA) = 0.12 W/cm(2), center frequency = 40 kHz, 90-second continuous wave) was applied on the sclera of New Zealand white rabbits for one to three cycles. Solution of fluorescent dextran (70 kDa) was placed above sclera during and after ultrasound application to assess transscleral transport of macromolecules. Amount of dextran delivered to vitreous was determined by detection of fluorescence. Visual function of ultrasound-treated rabbits was examined by full-field electroretinography (ffERG). The effect of ultrasound on ocular tissue structures was examined by binocular indirect ophthalmoscope (BIO) and histology. RESULTS: Repeated ultrasound resulted in increasing concentration of dextran, which was otherwise undetectable in the vitreous. Transscleral barrier against dextran transport was restored to original value at 2 weeks postultrasound treatment. Studies from ffERG suggested that electric responses from neural transmission of retinal cells are normal at 1 day, 7 days, and 14 days after ultrasound applications. BIO and histology revealed no structural abnormality in posterior ocular tissues after ultrasound treatment. CONCLUSIONS: Low-frequency ultrasound significantly enhanced the penetration of macromolecules via transscleral route. No undesirable side effects have been found for up to 2 weeks after ultrasound application. The study supports that sonication is a potentially safe and effective method to modulate transscleral barriers for delivering macromolecular therapeutics to posterior segment of the eye.

Specific uptake of folate-decorated triamcinolone-encapsulating nanoparticles by retinal pigment epithelium cells enhances and prolongs antiangiogenic activity.

We are proposing folate-decorated polymeric nanoparticles as carriers of poorly soluble drug molecules for intracellular and prolonged delivery to retinal pigment epithelium (RPE) cells. RPE is a monolayer of epithelial cells that forms the outer blood-retinal barrier in the posterior segment of the eye, and is also implicated in the pathology of, such as neovascularization in age-related macular degeneration (AMD). In this study, folate-functionalized poly(ethylene glycol)-b-polycaprolactone (folate-PEG-b-PCL) were synthesized for assembling into nanoparticles of ~130nm. These nanoparticles were internalized into ARPE-19 (human RPE cell line) via receptor-mediated endocytosis, and the cellular uptake was significantly higher than particles without folate modification. Triamcinolone acetonide (TA) was efficiently encapsulated (>97%) into the folate-decorated nanoparticles and was slowly released over a period of 4 weeks at pH 5.5 and 8 weeks at pH 7.4. The enhanced uptake and controlled release resulted in prolonged anti-angiogenic gene expression of RPE cells. In cell culture, the down-regulation of vascular endothelial growth factor (VEGF) and up-regulation of pigment epithelium derived factor (PEDF) lasted for at least 3 weeks. Unlike benzyl alcohol, the surfactant found in commercial formulation, folate-modified nanoparticles were non-toxic. Furthermore, TA became less cytotoxic by being encapsulated in the nanoparticles. Our findings suggest that folate-PEG-PCL nanoparticles are promising drug carriers for RPE targeting.