Microbubble-Assisted Ultrasound for Drug Delivery to the Retina in an Ex Vivo Eye Model.

Drug delivery to the retina is one of the major challenges in ophthalmology due to the biological barriers that protect it from harmful substances in the body. Despite the advancement in ocular therapeutics, there are many unmet needs for the treatment of retinal diseases. Ultrasound combined with microbubbles (USMB) was proposed as a minimally invasive method for improving delivery of drugs in the retina from the blood circulation. This study aimed to investigate the applicability of USMB for the delivery of model drugs (molecular weight varying from 600 Da to 20 kDa) in the retina of ex vivo porcine eyes. A clinical ultrasound system, in combination with microbubbles approved for clinical ultrasound imaging, was used for the treatment. Intracellular accumulation of model drugs was observed in the cells lining blood vessels in the retina and choroid of eyes treated with USMB but not in eyes that received ultrasound only. Specifically, 25.6 ± 2.9% of cells had intracellular uptake at mechanical index (MI) 0.2 and 34.5 ± 6.0% at MI 0.4. Histological examination of retinal and choroid tissues revealed that at these USMB conditions, no irreversible alterations were induced at the USMB conditions used. These results indicate that USMB can be used as a minimally invasive targeted means to induce intracellular accumulation of drugs for the treatment of retinal diseases.

Hyaluronic Acid-PEG-Based Diels-Alder In Situ Forming Hydrogels for Sustained Intraocular Delivery of Bevacizumab.

Retinal diseases are the leading cause of visual impairment worldwide. The effectiveness of antibodies for the treatment of retinal diseases has been demonstrated. Despite the clinical success, achieving sufficiently high concentrations of these protein therapeutics at the target tissue for an extended period is challenging. Patients suffering from macular degeneration often receive injections once per month. Therefore, there is a growing need for suitable systems that can help reduce the number of injections and adverse effects while improving patient complacency. This study systematically characterized degradable "in situ" forming hydrogels that can be easily injected into the vitreous cavity using a small needle (29G). After intravitreal injection, the formulation is designed to undergo a sol-gel phase transition at the administration site to obtain an intraocular depot system for long-term sustained release of bioactives. A Diels-Alder reaction was exploited to crosslink hyaluronic acid-bearing furan groups (HAFU) with 4 arm-PEG10K-maleimide (4APM), yielding stable hydrogels. Here, a systematic investigation of the effects of polymer composition and the ratio between functional groups on the physicochemical properties of hydrogels was performed to select the most suitable formulation for protein delivery. Rheological analysis showed rapid hydrogel formation, with the fastest gel formation within 5 min after mixing the hydrogel precursors. In this study, the mechanical properties of an ex vivo intravitreally formed hydrogel were investigated and compared to the in vitro fabricated samples. Swelling and degradation studies showed that the hydrogels are biodegradable by the retro-Diels-Alder reaction under physiological conditions. The 4APM-HAFU (ratio 1:5) hydrogel formulation showed sustained release of bevacizumab > 400 days by a combination of diffusion, swelling, and degradation. A bioassay showed that the released bevacizumab remained bioactive. The hydrogel platform described in this study offers high potential for the sustained release of therapeutic antibodies to treat ocular diseases.

The Effect of Microbubble-Assisted Ultrasound on Molecular Permeability across Cell Barriers.

The combination of ultrasound and microbubbles (USMB) has been applied to enhance drug permeability across tissue barriers. Most studies focused on only one physicochemical aspect (i.e., molecular weight of the delivered molecule). Using an in vitro epithelial (MDCK II) cell barrier, we examined the effects of USMB on the permeability of five molecules varying in molecular weight (182 Da to 20 kDa) and hydrophilicity (LogD at pH 7.4 from 1.5 to highly hydrophilic). Treatment of cells with USMB at increasing ultrasound pressures did not have a significant effect on the permeability of small molecules (molecular weight 259 to 376 Da), despite their differences in hydrophilicity (LogD at pH 7.4 from -3.2 to 1.5). The largest molecules (molecular weight 4 and 20 kDa) showed the highest increase in the epithelial permeability (3-7-fold). Simultaneously, USMB enhanced intracellular accumulation of the same molecules. In the case of the clinically relevant anti- C-X-C Chemokine Receptor Type 4 (CXCR4) nanobody (molecular weight 15 kDa), USMB enhanced paracellular permeability by two-fold and increased binding to retinoblastoma cells by five-fold. Consequently, USMB is a potential tool to improve the efficacy and safety of the delivery of drugs to organs protected by tissue barriers, such as the eye and the brain.

Ultrasound-Mediated Drug Delivery With a Clinical Ultrasound System: In Vitro Evaluation.

Chemotherapy efficacy is often reduced by insufficient drug uptake in tumor cells. The combination of ultrasound and microbubbles (USMB) has been shown to improve drug delivery and to enhance the efficacy of several drugs in vitro and in vivo, through effects collectively known as sonopermeation. However, clinical translation of USMB therapy is hampered by the large variety of (non-clinical) US set-ups and US parameters that are used in these studies, which are not easily translated to clinical practice. In order to facilitate clinical translation, the aim of this study was to prove that USMB therapy using a clinical ultrasound system (Philips iU22) in combination with clinically approved microbubbles (SonoVue) leads to efficient in vitro sonopermeation. To this end, we measured the efficacy of USMB therapy for different US probes (S5-1, C5-1 and C9-4) and US parameters in FaDu cells. The US probe with the lowest central frequency (i.e. 1.6 MHz for S5-1) showed the highest USMB-induced intracellular uptake of the fluorescent dye SYTOX™ Green (SG). These SG uptake levels were comparable to or even higher than those obtained with a custom-built US system with optimized US parameters. Moreover, USMB therapy with both the clinical and the custom-built US system increased the cytotoxicity of the hydrophilic drug bleomycin. Our results demonstrate that a clinical US system can be used to perform USMB therapy as efficiently as a single-element transducer set-up with optimized US parameters. Therefore, future trials could be based on these clinical US systems, including validated US parameters, in order to accelerate successful translation of USMB therapy.

Ultrasound and Microbubbles for the Treatment of Ocular Diseases: From Preclinical Research towards Clinical Application.

The unique anatomy of the eye and the presence of various biological barriers make efficacious ocular drug delivery challenging, particularly in the treatment of posterior eye diseases. This review focuses on the combination of ultrasound and microbubbles (USMB) as a minimally invasive method to improve the efficacy and targeting of ocular drug delivery. An extensive overview is given of the in vitro and in vivo studies investigating the mechanical effects of ultrasound-driven microbubbles aiming to: (i) temporarily disrupt the blood-retina barrier in order to enhance the delivery of systemically administered drugs into the eye, (ii) induce intracellular uptake of anticancer drugs and macromolecules and (iii) achieve targeted delivery of genes, for the treatment of ocular malignancies and degenerative diseases. Finally, the safety and tolerability aspects of USMB, essential for the translation of USMB to the clinic, are discussed.