ICG-mediated photodisruption of the inner limiting membrane enhances retinal drug delivery

Highlights

• ICG that is accumulated at the ILM can generate vapor nanobubbles upon irradiation with pulsed laser light.

• The formation and collapse of vapor nanobubbles can locally disrupt the ILM, rendering it more permeable for therapeutics.

• ILM photodisruption substantially enhances delivery of 120 nm-sized polystyrene & lipid nanoparticles into the bovine retina.

• ILM manipulation boosted the efficacy of mRNA-loaded lipid nanoparticles within the bovine retina by a factor of 5.

• This technology is also successful in ablating the complex and extraordinary thick human ILM.

Abstract

Many groundbreaking therapies for the treatment of blindness require delivery of biologics or cells to the inner retina by intravitreal injection. Unfortunately, the advancement of these therapies is greatly hampered by delivery difficulties where obstruction of the therapeutics at the inner limiting membrane (ILM) represents the dominant bottleneck. In this proof-of-principle study, we explore an innovative light-based approach to locally ablate the ILM in a minimally invasive and highly controlled manner, thus making the ILM more permeable for therapeutics. More specifically, we demonstrate that pulsed laser irradiation of ILM-bound indocyanine green (ICG), a clinically applied ILM dye, results in the formation of vapor nanobubbles which can disrupt the bovine ILM as well as the extraordinary thick human ILM. We have observed that this photodisruption allows for highly successful retinal delivery of model nanoparticles which are otherwise blocked by the intact ILM. Strikingly, this treatment is furthermore able of enhancing the efficacy of mRNA-loaded lipid nanoparticles within the bovine retina by a factor of 5. In conclusion, this study provides evidence for a light-based approach to overcome the ILM which has the potential to improve the efficacy of all retinal therapies hampered by this delivery barrier.

Introduction

Vision plays an essential role in every aspect and stage of our lives. Unfortunately, as estimated in the latest World Report on Vision by the WHO, vision impairment currently affects roughly 2.2 billion people, of which 36 million are genuinely blind [1]. Given that severe vision impairment has a detrimental impact on the quality of life of the affected individual along with its carers [2,3], substantial research by industry and academics is devoted to treating blinding diseases including age-related macular degeneration, glaucoma and inherited retinal dystrophies [4]. Many of these blinding pathologies originate within the retina, a remarkably complex light-sensitive tissue located at the back of the eye. Luckily, retinal gene therapy is advancing, as exemplified by the FDA approval of Voretigene Neparvovec, an AAV2 vector containing therapeutic DNA for treatment of Leber Congenital Amaurosis [5]. This vector is delivered by subretinal injection (Fig. 1B), an effective delivery route to target the retina since it evades many drug delivery barriers present within the eye [6]. Yet, while subretinal injection has undoubtedly proven its merit for delivering therapeutic genes to the outer retina, the interest in intravitreal (IVT) injection as delivery route is on the rise (Fig. 1A). The ease of execution, minimal invasiveness and the option for repeated administrations indeed renders IVT injection an ideal delivery route. The growing enthusiasm for IVT injection is furthermore fueled by a recent shift in attention from gene replacement therapy targeting the outer retina to other innovative strategies which require delivery to the inner retinal layers, including optogenetics [7], neuroprotection [8,9], treatment of inner retinal IRDs [10], retina regeneration [11] and cell reprogramming [12,13]. Unfortunately, many of these advanced therapies' pipelines are clogged due to delivery difficulties where the inner limiting membrane (ILM) represents the primary bottleneck. The ILM, forming the physical border between the vitreous and the retina, indeed greatly hampers the migration of IVT injected compounds into the retina. Its composition resembles that of a basement membrane with as its main components collagen IV, laminin, glycoproteins and heparan sulfate proteoglycans [14]. Strikingly, this ILM represents a strong barrier for virtually any type of retinal treatment [15], including non-viral vectors [16], viral vectors [15], stem cells [17], and some types of antibodies [15].

Conscious of this delivery hurdle and with the knowledge in mind that the ILM is non-essential in the adult eye, many research groups have attempted to disrupt the ILM with the aim of enhancing retinal delivery from the vitreal side. In addition, seeing that human ILM renewal by Müller cells is limited and extremely slow [ 13], one single treatment should suffice to guarantee a long-lasting effect. One strategy to overcome the ILM is enzymatic digestion, as introduced by Dalkara et al. [18] They observed a substantial increase in retinal AAV transduction in rats after mild digestion with the enzyme mix Pronase E. The same enzyme was successfully applied by Zhang et al. to augment neurite engraftment of transplanted human embryonic stem cell-derived retinal ganglion cells into the mouse retina [17]. Another strategy to evade the ILM is subILM injection, as evaluated by Gamlin et al., which was efficient in enhancing AAV delivery to the primate retina [19]. Likewise, Takahashi et al. observed that surgical ILM peeling substantially increased viral transduction of the inner retina in monkeys [20]. Although these methods clearly demonstrate the power of ILM manipulation or evasion, enzymatic digestion is difficult to control and both ILM peeling and subILM injections involve invasive surgery [21]. Besides, subILM injection does not allow widespread retinal expression, which is regarded as a major advantage of IVT delivery [22].

In this study, we explore an innovative light-based approach to locally ablate the ILM in a minimally invasive and highly controlled manner, thus making the ILM more permeable for therapeutics. This approach, called photoporation, is based on the use of extremely short but powerful laser pulses (<7 ns) which have the capacity to locally generate nanoscopic bubbles (‘vapor nanobubbles’ or VNBs) when photothermal nanoparticles are irradiated. The mechanical force arising from expanding and collapsing VNBs, formed by the evaporation of water surrounding the photothermal entities, has the capacity to compromise nearby biological structures. While photoporation was initially investigated to transiently rupture cellular membranes, as intensively investigated by our group in recent years [[23], [24], [25], [26], [27]], its applicability has extended to other uses as well such as enhancing delivery of therapeutics in bacterial biofilms [28,29], ablation of tissue [30], and destroying collagen-based floaters in the vitreous [31,32]. Due to the extremely short lifetime of VNBs (10–100 ns) and the thermal insulating property of vapor, diffusion of heat to the environment is negligible. Therefore, VNBs are ideally suited to cause local disruptions in their surroundings without causing thermal damage to nearby cells or tissues. Conveniently, the extent of local disruption is tunable by adjusting the laser pulse energy which affects the size of VNB that will be formed.

In the majority of the cases, photoporation is carried out making use of photothermal particles such as gold nanoparticles (AuNPs) owing to their unique plasmonic properties [27,31]. Yet, recent reports on their toxicity along with their fragmentation upon laser irradiation have inspired researchers to look for more biocompatible alternatives [25,33,34]. Interestingly, our research group has recently discovered that the organic dye indocyanine green (ICG) is very well suited to generate VNBs upon illumination with pulsed laser light, at least in those places where ICG locally accumulates [35]. ICG is an FDA-approved dye with a long history of clinical use in several fields, including ophthalmology. In 2000, it was discovered that ICG could brightly stain the – otherwise transparent – human ILM, a finding which greatly facilitated challenging vitreoretinal surgeries [36]. Despite the fact that other ILM dyes have been discovered in the meantime, the use of ICG remains widespread. As an example, 70% of U.S. retina specialists use ICG during surgery [37]. Initially, concentrations up to 5 mg/ml were commonly applied, yet emerging debates regarding ICG's toxicity have led to the use of concentrations below 1 mg/ml [38,39]. One of ICG's assets, rendering it popular for in vivo imaging, is its high absorbance in the NIR range (600–900 nm), a relatively transparent window for biological tissues [40,41]. In addition, the photothermal properties of ICG have been widely investigated, mainly for photodynamic therapy in context of cancer treatment [42]. In summary, the combination of ICG's inherent affinity to the ILM along with its photothermal qualities, persuaded us to assess its potential as photosensitizer for photodisruption of the ILM.

As illustrated in Fig. 2, we hypothesized that ICG could generate VNBs when applied to the ILM surface and illuminated by an extremely short picosecond laser pulse (2 ps) of 800 nm. The mechanical forces induced by VNBs could then compromise the integrity of the ILM, allowing therapeutics to enter the retina. In this way, our approach would overcome the ILM by combining conventional methods already available in the clinic, i.e. IVT injection and laser application in the eye, with the innovative concept of VNB creation by a picosecond laser and ICG.

In this study, we provide proof of principle for ICG-mediated ILM photodisruption in ex vivo bovine retinal explants. To this end, we evaluated the integrity of the ILM and retinal viability following photodisruption in function of varying laser energy and ICG concentration. The positive impact of ILM photodisruption on retinal delivery was furthermore demonstrated with model particles (FluoSpheres™) as well as state-of-the-art lipid nanoparticles (LNPs). In addition, we verified our observations in human explants while critically reflecting on the species-specific differences in ILM morphology between the bovine and human retina.