Microbubble-assisted ultrasound for retinal drug delivery

The objective of this thesis was to investigate the potential of ultrasound and microbubble (USMB) assisted drug delivery as a means to treat retinal diseases. Retinal diseases such as age-related macular degeneration and diabetic retinopathy are typically treated with intravitreal injection of anti-vascular endothelial growth factor (anti-VEGF) drugs. Owing to fast elimination of drugs from the vitreous humor, intravitreal injections need to be repeated (bi)monthly, which is often related to side effects. Targeting the retina from the blood circulation could be a less invasive alternative to intravitreal injections. However, intravenously administered drugs need to bypass the blood-retina barrier (BRB) before they have access to the retina. Thus, a method that enables disruption of the BRB in a safe and reversible manner could potentially be combined with intravenously administered drugs. The potential of USMB to improve ocular drug delivery has been explored worldwide. This thesis reviews the preclinical studies that examined this and discusses the aspects related to safety of USMB for drug delivery in the eye. The efficacy of USMB in the transport of molecules with different physicochemical characteristics across an epithelial cell barrier was studied. Molecules varied in molecular weight (182 Da to 20 kDa) and hydrophilicity (LogD at pH 7.4 from 1.5 to highly hydrophilic). Different ultrasound pressures were used for the USMB treatment (Pneg 0.3-0.7 MPa). USMB did not alter the permeability of small molecules (molecular weight 259 to 376 Da) at any of the ultrasound pressures used, despite their differences in hydrophilicity. On the contrary, for the two large molecules (molecular weight 4 and 20 kDa) permeability was significantly increased (3-7-fold) at the two highest ultrasound pressures . At the same time, intracellular accumulation of the same large hydrophilic molecules was facilitated by USMB at Pneg of 0.7 MPa. Furthermore, the effect of USMB on the permeability of the barrier was investigated using a clinically relevant molecule (anti-CXCR4 nanobody, molecular weight 15 kDa) as a model drug for the treatment of retinoblastoma. USMB doubled the permeability of nanobody across the cell barrier and increased binding to retinoblastoma cells by five-fold. To study the efficacy of USMB in a more physiologically representative system than an in vitro model, an experimental platform using arterially perfused ex vivo porcine eyes was developed. Using ex vivo eyes, the effect of USMB on the porcine retina was investigated. A clinical imaging ultrasound system and commercially available microbubbles were used for the treatment at two different mechanical indexes (MI of 0.2 and 0.4). Intracellular accumulation of model drugs (molecular weight of 0.6-20.0 kDa) was observed in eyes treated with USMB but not in eyes that received ultrasound only. Intracellular accumulation was observed in cells lining the blood vessels in the retina and choroid. The encouraging results obtained in this thesis showed a first promising step towards implementation of USMB-assisted drug delivery in the clinic to improve the cure of retinopathies.