High-Frequency Ultrasound Activation of Perfluorocarbon Nanodroplets for Treatment of Glaucoma

Abstract

Elevated intraocular pressure is the most prevalent risk factor for initiation and progression of neurodegeneration in glaucoma. Ocular hypertension results from increased resistance to aqueous fluid outflow caused by reduced porosity and increased stiffness of tissues of the outflow pathway. Acoustic activation and resulting bioeffects of the perfluorocarbon nanodroplets (NDs) introduced into the anterior chamber of the eye could potentially represent a treatment for glaucoma by increasing permeability in the aqueous outflow track. To evaluate the potential of NDs to enter the outflow track, 100-nm diameter perfluoropentane NDs with a lipid shell were injected into the anterior chamber of ex vivo pig eyes and in vivo rat eyes. The NDs were activated and imaged with 18 MHz and 28 MHz linear arrays to assess their location and diffusion. NDs in the anterior chamber could also be visualized using optical coherence tomography. Because of their higher density with respect to aqueous humor, some NDs settled into the iridocorneal angle where they entered the outflow pathway. After acoustic activation of the NDs at the highest acoustic pressure, small gas bubbles were observed in the anterior chamber. After two days, no acoustic activation events were visible in the anterior chamber of the rats and their eyes showed no evidence of inflammation.

I. Introduction

A. Glaucoma

Glaucoma refers to a group of chronic and progressive ocular pathologies of multifactorial etiology, but all characterized by degeneration of optic nerve axons and death of retinal ganglion cells. Glaucoma is the leading cause of blindness worldwide and the most common cause of vision loss in the United States. Although prevalence varies by race and region, primary open-angle glaucoma (POAG) is the most common form of glaucoma worldwide and is estimated to affect 57.5 million people globally [1]

Elevated intraocular pressure (IOP) represents the major risk factor for glaucoma and is currently the only modifiable such factor. Ocular hypertension results from an imbalance between aqueous fluid production by the ciliary body epithelia in the posterior chamber and drainage via the corneoscleral angle in the anterior chamber.

Aqueous fluid drains from the anterior chamber (AC) through the trabecular meshwork (TM), the juxtacanalicular connective tissue, the endothelial lining of the Schlemm’s canal (SC), the SC itself, the collecting channels, aqueous veins, and ultimately the episcleral venous system. Collectively, this flow path is known as the ‘conventional outflow pathway’ (Fig. 1). It has long been recognized [2] that the elevated IOP characteristic of POAG arises from increased resistance to aqueous outflow at some point along the outflow pathway.

Fig. 1.

Diagrammatic depiction of anterior segment of the eye. Aqueous humour formed by the ciliary processes passes through the pupil into the anterior chamber. Aqueous passes through the pores of the trabecular meshwork (TM) and enters the Schlemm’s canal (SC).

The TM, through which aqueous enters the conventional outflow pathway, is a porous structure, with uveal meshwork pores averaging 42.6 μm in diameter in its proximal regions and 8.9 μm in the deeper corneoscleral meshwork [3], which is sometimes referred to as the juxtacanalicular connective tissue. The TM is ~100 μm thick overall. Calculations of flow resistance based on the dimensions of the flow passages in the meshwork indicated that the normal TM should have negligible flow resistance [4]. Nevertheless, the TM has been implicated in increased outflow resistance in POAG.

The SC is a flattened circumferential channel in proximity to the TM (Fig. 1). Using optical coherence tomography (OCT), Shi [5] reported an in vivo SC meridional diameter of 266.96 ± 49.55 μm. Yan [6], using 80 MHz ultrasound biomicroscopy, reported a long cross-sectional diameter of 233.0 ± 34.5 μm and a short diameter of 44.5 ± 12.6 μm.

Recent studies have focused on the SC and the juxtacanalicular connective tissue as the principal sites of outflow resistance. As long ago as 1992, reduced pore density in the inner wall endothelial lining of the SC in POAG versus normal eyes was reported [7]. More recently, Johnson et al. reported that glaucomatous eyes exhibit only 20% of the pore density of normal eyes [8]. Johnson stated that “The locus of the outflow resistance, both in the normal eye and the glaucomatous eye, is thought to arise either in the endothelial lining of the SC, or very near to this location [9]. Decreased porosity and increased stiffness have also been reported in the juxtacanalicular connective tissue and endothelial cells of the SC in glaucomatous eyes, [10], [11]. Increased stiffness has been observed in the inner-wall tissue of the SC in glaucomatous eyes, where the SC endothelial cells and underlying extracellular matrix reside [12].

IOP-lowering medical therapy is the first line approach for management of POAG [13]. Current pharmacologic approaches to glaucoma management include several families of topically applied eye drops that either reduce aqueous production or improve outflow: Pharmacologic treatments, however, are associated with side-effects including ocular toxicity [14] and surface disease [15]. Also, medications must be taken for life, and hence compliance issues are a major problem.

Surgical interventions are in general resorted to when pharmacologic approaches are unsuccessful in adequately controlling IOP. A variety of procedures are available, including shunts and surgical approaches targeting the SC, but success is variable.

B. Therapeutic Potential of Acoustic Cavitation in POAG

Given our current understanding of the pathogenesis of POAG, methods that directly target the increased resistance to outflow stemming from the decreased porosity and increased stiffness of the SC and juxtacanalicular tissues would be of great significance.

Gas-filled microbubbles of 1-5 μm diameter, typically with protein or phospholipid shells, have a long history as circulating ultrasound contrast agents [16]. These microbubbles scatter acoustic energy but also undergo oscillations that can initiate acoustic cavitation. Acoustic cavitation can be divided into (i) stable cavitation at lower ultrasound intensities where the microbubble diameter continuously resonates in concert with ultrasound wave rarefaction and compression cycles and (ii) inertial cavitation at higher ultrasound intensities characterized by microbubble implosion/destruction, which will result in much stronger biophysical effects. Biologic effects of cavitation may result from several mechanisms, including direct impingement of the bubble wall with tissue, microstreaming, microjetting and shock waves, among other mechanisms [17].

Sonoporation of the SC and TM would seem a natural application of this methodology but is not directly applicable for several reasons; Firstly, the TM and the SC are themselves not vascular, and hence introduction of microbubbles into the general circulation is unlikely to be effective. Secondly, microbubbles introduced into the AC will float because they are less dense than aqueous humor, and this prevents them from maintaining contact with the TM or entering the SC. Thirdly, the diameter of microbubbles is not much smaller than corneoscleral TM pores, which would impede diffusion into the SC. Finally, while inertial cavitation will destroy circulating intravascular microbubbles, they are rapidly replenished as new microbubbles enter from the general circulation; this would not be the case where bubbles are confined to the AC or outflow pathways.

C. Perfluorocarbon (PFC) Nanodroplets

PFC NDs address the above limitations. Lipid-coated PFC NDs with diameters of a few hundred nanometers have been developed to serve as a diagnostic ultrasound contrast agent [18] and as cavitation nuclei in therapeutic applications [19]. NDs consist of a liquid PFC core that can be phase-shifted into gas in a process termed acoustic droplet vaporization. NDs are advantageous with respect to traditional gas-filled microbubble contrast agents by virtue of their order-of-magnitude smaller diameter which facilitates penetration into tissues. Acoustically activated NDs have been shown to permeabilize blood vessels [20]. Taking advantage of the ability of NDs to extravasate within the leaky vasculature of tumors, perfluorohexane (PFH) NDs (boiling point 56°C) in combination with high intensity focused ultrasound was demonstrated to enhance destruction of ovarian tumors in a mouse model [21].

PFC NDs offer multiple advantages for POAG treatment: they can be activated to form cavitating microbubbles at near-diagnostic ultrasound intensities; they can be designed to recondense back to a liquid state, so bubbles don’t persist; their small dimension facilitates passage thru pores in the TM; their relatively high density prevents floating to coat the inner cornea. These properties tend to keep the NDs in suspension and/or (with the eye facing upwards) settling on the surface of the iris, lens or entering the iridocorneal angle rather than floating to coat the inner wall of the cornea as would a microbubble. Thus, ultrasound can be used for simultaneous visualization and targeting of treatment in the angle region.

One disadvantage of PFH NDs is the potential of unwanted thermal and mechanical effects in nearby tissues because of the high peak negative pressure required to induce a phase transition. To address this limitation, Liu described NDs with a perfluoropentane (PFP) core [22], enabling phase transition with lower peak negative pressures. Note that although the boiling point of PFP is 29°C, PFP NDs do not vaporize at body temperature due to the Laplace pressure exerted on the PFC core at the ND lipid boundary [23].

PFC liquids have been used as a short-term vitreous substitute in retinal detachment surgery and other ophthalmic surgical procedures for decades [24]. Here, the ND preparation is an emulsion of lipid-coated nanoparticles of 100-200 nm diameter rather than a liquid.

Circulating intravascular NDs can extravasate into tissues because of their small diameter. In this study, we investigate transport of NDs from the AC into the outflow pathway, bringing them into contact with the tissues known to have reduced porosity and increased stiffness in POAG.

II. Methods

A. Formulation of NDs

In this investigation, we used NDs with a PFP core and a lipid shell [25]. PFP is particularly useful because it is stable at physiological temperature and able to be converted into microbubbles at clinically relevant acoustic intensities, i.e., at levels occurring in diagnostic ultrasound systems.

NDs were fabricated to have a lipid shell consisting of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC, CAS # 816-94-4, Avanti Polar Lipids, Alabaster, AL, USA) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000] (ammonium salt) (DSPE-PEG2000, CAS # 474922-77-5, Avanti Polar Lipids). The fabrication process started by dissolving lipids in chloroform transferring to a glass vial. By gently evaporating the chloroform, a thin lipid film was formed on the side of the vial. The film was then hydrated with an aqueous buffer containing 80% (v/v) phosphate buffered saline, 10% (v/v) propylene glycol, and 10% (v/v) glycerol such that the final lipid concentrations were 6.3 mM DSPC and 0.4 mM DSPEPEG2000.

100 μL of PFP and 5 mL of lipid solution were sonicated (Branson 450, Emerson, St. Louis, MO, USA) to form a course emulsion. This coarse emulsion was processed using a high-shear, bench-top homogenizer (LV1, Microfluidics, Westwood, MA, USA) to further reduce the particle size distribution. Larger NDs were removed by spinning the emulsion for 1 minute at 2000 relative centrifugal force to pellet larger NDs and the supernatant, containing smaller NDs, was isolated and used for experiments. NDs were stored at 4 °C for up to a month. ND sizing was performed with a laser diffraction particle sizing analyzer (LS 13 320, Beckman Coulter, Inc. Brea, CA, USA).

B. Ultrasound Imaging and Activation of NDs

The ultrasound system was used in a dual imaging mode where a standard B-mode image was acquired and then an activation/imaging sequence was initiated. The activation/imaging data were overlaid on the B-mode image to capture the anatomical information and the co-registered regions of ND activation.

The 128-line B-mode images were derived from a standard line-by-line sequence using a 28-element (2.8 mm) sliding transmit/receive aperture centered on each image line, yielding a F=2.86 focal ratio at the 8 mm focal depth. The B-mode sequence was used in a real-time mode to locate the region of interest prior to initiating the activation sequence. The B-mode transmit pressures were lower than the activation sequence due to the smaller transmit aperture.

Once the B-mode image displayed the tissue region of interest, the ND activation/imaging sequence was initiated. This consisted of a line-by-line transmit/receive sequence using a variable aperture. At line 1 (leftmost), the aperture had 64 elements. This increased by 1 until reaching line 64 (center line) where all 128 elements were used and then decreased back to 64 at line 128 (rightmost elements). This created a tightly focused beam for ND activation within a focal zone that was ~0.5 mm long in the depth axis. The variation in width of the transmit aperture (from 64 to 128 elements) with line position resulted in the F-number ranging from 1.25 to 0.625 where the maximum transmit pressure occurred at the center of the image, tapering off to either side. This sequence imaged ND activation events by capturing the scattering of the newly formed transient microbubbles and superimposing on the B-mode image.

The activation maps could be formed with a variable number of depth-based focal zones, centered around a depth of 8 mm. The final activation map was stitched together from all the focal zones with the zones assembled by first sweeping across all the image lines at one depth before moving to the next depth. In addition, the system could be programmed to only activate/image within a defined central region of the image. After applying an intensity threshold to the activation image data, the activation map was overlaid as a heat map on top of the greyscale B-mode image acquired prior to the start of the activation sequence.

A total of 100 activation frames were captured at a frame rate of 100-500 Hz, depending on the number of focal zones, and the raw received channel data saved and processed in real-time. A spatiotemporal clutter filter based on singular-value decomposition (SVD) was used to separate ND activation regions from tissue. A mean intensity image was then produced using the 100-frame stack of SVD-filtered images and overlaid as a heat map onto the B-mode imaging for real-time visualization of ND activation.

C. Experimental Procedures

We introduced 0.1 ml of PFP NDs into the AC of ex vivo pig eyes using a 30g needle. Pig eyes are anatomically similar and of comparable dimension to human eyes. The eye was submerged in 37°C normal saline solution and imaged using the 18-MHz linear array at a 20-V excitation with 1, 3 and 5 focal zones. The AC was cannulated with a 30g needle attached to an IV line with a pressure transducer and a normal saline bag was elevated to bring the IOP to 15 mmHg, which is an approximately normal in vivo IOP.

We also introduced NDs into the AC of three in vivo Sprague Dawley rats weighing ~250g. Although the rat eye is far smaller than the human eye, its outflow anatomy is similar, and rats are widely used to experimentally model human glaucoma. Using a Zeiss operating microscope and micromanipulator (M3301r, World Precision Instruments, Sarasota, FL), a 33g beveled needle attached to a Hamilton syringe was introduced into the AC of the anesthetized rat, and ~25 μl of NDs were injected. Anterior chambers were imaged with the 28-MHz probe at a series of intensities. After the ultrasound imaging, the eyes were imaged by OCT. After two days, the rats were reexamined for evidence of inflammation.

D. Acoustic Activation and Imaging

The acoustic pressure at the transducer focus was measured using a calibrated 40-μm diameter needle hydrophone (Precision Acoustics, Inc., Dorset, UK) positioned in the elevation focal plane at the center of the array aperture. We excited the array from 10V to the maximum excitation voltage of 40V, varying the number of transmit elements.

III. Results

The size distribution of NPs is presented in Fig. 2.

Fig. 2.

Distribution of NDs by radius and volume (assuming spherical shape).

A plot of peak negative pressure as a function of voltage and number of transmit elements for the 28 MHz probe is provided in Fig. 3.

Fig. 3.

Peak negative pressure and mechanical index for a focused (F=0.625) transmit to ~8 mm on 28 MHz probe.

Images of activated NDs in the ex vivo pig eye are shown in Figs. 4 and 5. With a single focus, the activation is in just one plane, but by increasing the number of focal zones, NDs can be activated over a greater depth of field. Of note is the presence of a flattened feature in proximity to the iridocorneal angle, which would be consistent with the presence of NDs in the SC.

Fig. 4.

NDs in anterior chamber of ex vivo pig eye. Left: NDs activated in the focal plane of the probe in the central AC. Right: NDs after diffusion into the angle and outflow path. C=cornea, AC=anterior chamber, L=lens, S=sclera, CB=ciliary body. Arrow points to flattened space consistent with SC.

Fig. 5.

NDs in ex vivo pig eye 30 minutes after intracameral injection. Anterior chamber is cannulated and IOP brought to 15 mmHg. Imaging was performed with the 18 MHz probe using 5 focal depths, enabling activation and visualization of NDs throughout the anterior chamber. Note some NDs settling on surface of lens and iris due to their relatively high density with respect to aqueous. NDs appear to have entered outflow pathway (arrow).

Fig. 6 demonstrates, in the in vivo rat eye, the increased likelihood of activation as the acoustic pressure increased. At 20V, the acoustic pressure was sufficient to activate some fraction of NDs, but a large portion of NDs were still below the activation threshold. As the excitation increased to 30V and then 35V, the fraction of NDs that were activated greatly increased. At the 30V excitation voltage threshold for extensive activation, the mechanical index (MI) was 1.18.

Fig. 6.

NDs in rat anterior chamber in vivo imaged with 28 MHz probe. Activation transmits were limited to the central 2 mm of the scan. a) Greyscale B-mode image and SVD-filtered images of activated NDs at b) 20V, c) 30V and d) 35V excitation.

Fig. 7 demonstrates OCT images of the anterior segment and angle about an hour after ND injection in the rat eye. The images demonstrate optical backscatter from NDs present in the AC, and especially in the region of the iridocorneal angle.

Fig. 7.

In vivo OCT images of full anterior segment (top, 1.5 mm in depth x 6 mm in width) and temporal angle (below, 1.5 x 1.5 mm) of rat eye one hour after intracameral injection of NDs. C=cornea, AC=anterior chamber, L=lens, I=iris, CB=ciliary body.

Two days after injection, rats appeared healthy with no evidence of an ocular inflammatory response. However, a few small gas bubbles were observed in the AC.

IV. Discussion

In these experiments, we demonstrated that PFP NDs injected into the anterior chambers of ex vivo pig eyes and in vivo rat eyes could be activated with 18- and 28-MHz focused ultrasound at intensities comparable to diagnostic levels. Nevertheless, at the 30V activation threshold for the 28 MHz probe, an MI of 1.18 was found, which substantially exceeds the FDA limit of 0.23 for diagnostic ultrasound imaging of the eye (the most stringent criteria for any tissue region). However, since the envisioned application is therapeutic rather than diagnostic, this consideration is not a significant limitation. Indeed, having a therapeutic agent that does not activate at diagnostic levels may be advantageous.

NDs in the pig eye accumulated in the angle and entered the SC. NDs introduced into the in vivo rat eye were readily imaged and well-tolerated by rats over a two-day period.

We noted the presence of small gas bubbles in the AC of both ex vivo pig eyes and in vivo rat eyes following experiments performed at the highest excitation voltage. We anticipate that this effect would be reduced at lower acoustic pressures where only stable cavitation is produced [26]. We would expect the NDs to leave the eye via the outflow channels and venous circulation, but persistence will need to be verified in future studies. Note that PFP NDs introduced into the venous system have a reported persistence of only minutes [27].

Although OCT provides higher spatial resolution than ultrasound, OCT images only demonstrate optical backscatter from NDs suspended in the AC. OCT does not activate NDs to produce increased contrast in OCT images and hence does not allow their differentiation from tissue optical backscatter in and around the aqueous outflow pathway. Although OCT will not activate PFC NDs to produce a therapeutic effect, OCT will be useful in the future for in vivo evaluation of bioeffects on tissues of the outflow pathway and anterior segment.

Besides POAG, various forms of secondary open-angle glaucoma, including exfoliative glaucoma and pigmentary glaucoma, may also benefit from this treatment modality.

Going forward, we hypothesize that activation of NDs introduced into the outflow pathway may act to improve outflow by increasing the permeability of the SC and juxtacanalicular tissues which are known to suffer increased stiffness and decreased porosity in POAG. Quantitative acoustic microscopy may be useful in demonstrating biomechanical changes in tissues of the outflow pathway [28, 29]. In order to test this hypothesis, the longevity of effectiveness and long-term tolerance, in vivo studies of IOP and outflow facility in glaucoma models, first in the rat and later in non-human primates, will be necessary.

V. Conclusion

In these experiments, we demonstrated that ~100-nm diameter PFP NDs introduced into the AC of the eye will extravasate into the outflow pathway. Future studies will investigate the effect of ultrasound-induced cavitation of NDs present in the SC and other tissues of the outflow pathway in reducing outflow resistance as a means for reducing intraocular pressure in open angle glaucoma.

Biographies

Ronald H. Silverman received his M.S. (Bioengineering) and Ph.D. (Computer Science) degrees from Polytechnic University in Brooklyn, NY in 1979 and 1990, respectively. His primary research focus is the development and application of ultrasound systems and signal-processing methodologies for diagnostic imaging of the eye. After 28 years at the Weill Cornell Medical Center in New York, NY, he joined the Department of Ophthalmology of the Columbia University Medical Center in 2010, where he is now Professor of Ophthalmic Science. Dr. Silverman is a Senior Member of IEEE, a Fellow of the American Institute of Ultrasound in Medicine (AIUM), and a Fellow of the American Institute for Medical and Biological Engineers.

Raksha Urs was born in India. She received the Bachelor of Engineering degree from the University of Mysore in India in 1996 and the MS and Ph.D in Biomedical Engineering from the University of Miami in 2006 and 2010. Her doctoral work was at the Bascom Palmer Eye Institute in Florida where she designed and developed ultrasound imaging and analysis techniques for lens and ciliary muscle dynamics during accommodation. She is an Associate Research Scientist at Columbia University Irving Medical Center in the Department of Ophthalmology where she is developing methods for imaging ocular blood-flow and perfusion using Ultrasound Plane-Wave imaging.

Mark Burgess (M’20) was born in Cincinnati, OH in 1986. He received a B.S. degree in biomedical engineering from the University of Cincinnati in 2010. He received M.S. and Ph.D. degrees in Mechanical Engineering from Boston University in 2013 and 2016, respectively. His thesis work focused on the control of acoustic cavitation for efficient intracellular delivery of macromolecules. Dr. Burgess joined the Lizzi Center for Biomedical Engineering at Riverside Research in 2020 where he is a member of the research staff. His current interests are contrast-enhanced ultrasound imaging, ultrafast plane-wave imaging, and image-guided drug delivery to the brain with focused ultrasound.

Jeffrey A. Ketterling (Senior Member’11) was born in Seattle, WA. He received the B.S. degree in electrical engineering from the University of Washington, Seattle, WA, in 1994. He received the Ph.D. degree in mechanical engineering from Yale University, New Haven, CT, in 1999. His thesis focused on experimental studies of phase-space stability in single bubble sonoluminescence.

Dr. Ketterling joined the Lizzi Center for Biomedical Engineering at Riverside Research in 1999 and is currently the Associate Research Director. He serves as a principal investigator for programs supported by the National Institutes of Health that deal with high-frequency annular arrays for small-animal and ophthalmic imaging, acoustic contrast agents for microcirculation imaging, vector-flow imaging of blood-flow patterns in animal models, high-speed plane-wave imaging and hydrophone arrays for characterizing the instantaneous acoustic fields of lithotripters.

Dr. Ketterling was the Technical Chair for the Biomedical Acoustics Committee of the Acoustical Society of America from 2008-2011 and the Chair of the Group 1 Medical Ultrasonics Tech. Program Committee of the IEEE International Ultrasonics Symposium from 2019-2021.

Gülgün Tezel obtained her M.D. degree in 1983, completed her ophthalmology residency in 1989, and continued her career as a glaucoma researcher scientist. In 1999, she joined the research faculty at Washington University, in St. Louis, MO, and then established her laboratory at the University of Louisville in Louisville, KY in 2002. In 2014, her laboratory was transferred to the Edward S. Harkness Eye Institute at the Columbia University in New York, NY, where she is currently a Professor of Ophthalmic Sciences. Dr. Tezel’s research is focused on cellular and molecular processes of retinal ganglion cell death, neuron-glia interactions, and immune/inflammatory responses to identify new treatment targets and biomarkers for glaucomatous neurodegeneration. Dr. Tezel has received many professional awards, including the Research to Prevent Blindness Sybil B. Harrington Scholars Award and the ARVO Fellowship. She currently serves on the Scientific Advisory Board of the Glaucoma Foundation and the Editorial Boards of the scientific journals in vision research, Progress in Retinal and Eye Research, and Investigative Ophthalmology & Visual Science.