Gold Nanoparticles for Retinal Molecular Optical Imaging.

The incorporation of gold nanoparticles (GNPs) into retinal imaging signifies a notable advancement in ophthalmology, offering improved accuracy in diagnosis and patient outcomes. This review explores the synthesis and unique properties of GNPs, highlighting their adjustable surface plasmon resonance, biocompatibility, and excellent optical absorption and scattering abilities. These features make GNPs advantageous contrast agents, enhancing the precision and quality of various imaging modalities, including photoacoustic imaging, optical coherence tomography, and fluorescence imaging. This paper analyzes the unique properties and corresponding mechanisms based on the morphological features of GNPs, highlighting the potential of GNPs in retinal disease diagnosis and management. Given the limitations currently encountered in clinical applications of GNPs, the approaches and strategies to overcome these limitations are also discussed. These findings suggest that the properties and efficacy of GNPs have innovative applications in retinal disease imaging.

Age differential response to bevacizumab therapy in choroidal neovascularization in rabbits.

Choroidal neovascularization (CNV) in young rabbits has been shown to have a rapid, robust response after treatment with bevacizumab, an anti-vascular endothelial growth factor (VEGF) medication. This investigation evaluates an age differential response to bevacizumab in older populations of rabbits using multimodal high resolution molecular imaging. Young (4 months old) and life span (14 months old) rabbits were given subretinal injections of Matrigel and VEGF to produce CNV. All CNV rabbit models were then treated with a bevacizumab intravitreal injection. Rabbits were then monitored longitudinally using photoacoustic microscopy (PAM), optical coherence tomography (OCT), color photography, and fluorescence imaging. Chain-like gold nanoparticle clusters (CGNP) conjugated with tripeptide arginylglycylaspartic acid (RGD) was injected intravenously for molecular imaging. Robust CNV developed in both young and old rabbits. After intravitreal bevacizumab injection, fluorescence signals were markedly decreased 90.13% in the young group. In contrast, old rabbit CNV area decreased by only 10.56% post-bevacizumab treatment. OCT images confirmed a rapid decrease of CNV in the young group. CGNPs demonstrated high PAM signal in old rabbits and minimal PAM signal in young rabbits after bevacizumab, indicating CNV regression. There is a significant difference in response to intravitreal bevacizumab treatment between young and old rabbits with CNV which can be monitored with multimodal molecular imaging. Old rabbits demonstrate significant persistent disease activity. This represents the first large eye model of persistent disease activity of CNV and could serve as the foundation for future investigations into the mechanism of persistent disease activity and the development of novel therapies.

Retinal safety evaluation of photoacoustic microscopy.

Photoacoustic microscopy (PAM) has significant potential as a promising diagnostic method for eye diseases and can provide anatomic and functional information of the retinal and choroidal vasculature. However, there are no FDA-approved PAM systems for ophthalmic imaging. In this study, a comprehensive safety evaluation was performed to evaluate the safety of PAM retinal imaging and whether PAM causes damage to retinal structure or function in rabbit eyes. 12 Dutch-Belted pigmented rabbits received photoacoustic imaging to 57% of the retinal surface area with a laser energy of 5% of the ANSI safety limit for five consecutive days and followed before imaging and 3 days, 1, 2, 3, and 4 weeks post imaging. Retinal morphologic analyses using slit lamp examination, fundus photography, red free, FA, FAF, ICGA, and OCT showed no retinal hemorrhage, edema, detachment, vascular abnormalities, or pigmentary abnormalities in the retina or choroid after PAM imaging. Full-field ERG analysis showed no significant difference in scotopic or photopic a- and b-wave amplitudes or implicit times between the control and experimental eyes over time (n = 6, P values > 0.05). Retinal ultrastructural evaluation using TEM showed normal structure of organelles and nuclei, and no significant loss of cells after PAM. TUNEL assay showed no evidence of cells apoptosis in retina. Retinal histopathology indicated that the architecture and thickness of the retinal layers was well preserved in all experimental eyes. A positive control at 500% of the ANSI limit demonstrated significant damage. The comprehensive retinal safety evaluation demonstrated no damage to retinal structure or function for 4 weeks after PAM imaging in rabbits.

High Resolution Multimodal Photoacoustic Microscopy and Optical Coherence Tomography Visualization of Choroidal Vascular Occlusion.

Photoacoustic microscopy is a novel, non-ionizing, non-invasive imaging technology that evaluates tissue absorption of short-pulsed light through the sound waves emitted by the tissue and has numerous biomedical applications. In this study, a custom-built multimodal imaging system, including photoacoustic microscopy (PAM) and optical coherence tomography (OCT), has been developed to evaluate choroidal vascular occlusion (CVO). CVO was performed on three living rabbits using laser photocoagulation. Longitudinal imaging of CVO was obtained using multiple imaging tools such as color fundus photography, fluorescein angiography, indocyanine green angiography (ICGA), OCT, and PAM. PAM images were acquired at different wavelengths, ranging from 532 to 700 nm. The results demonstrate that the CVO was clearly observed on PAM in both two dimensions (2D) and 3D with high resolution longitudinally over 28 days. In addition, the location and margin of the CVO were distinguished from the surrounding choroidal vasculature after the injection of ICG contrast agent. PAM imaging was achieved using a laser energy of approximately 80 nJ, which is about half of the American National Standards Institute safety limit. The proposed imaging technique may provide a potential tool for the evaluation of different chorioretinal vascular disease pathogeneses and other biological studies.

Real-time OCT guidance and multimodal imaging monitoring of subretinal injection induced choroidal neovascularization in rabbit eyes.

Choroidal neovascularization (CNV) is a major cause of vision loss that consists of abnormal growth of new blood vessels from the choroidal vasculature. High resolution in vivo imaging of animal models is essential to better elucidate and conduct research on CNV. This study evaluates a novel multimodal imaging platform combining optical coherence tomography (OCT) and photoacoustic microscopy (PAM). Using real-time OCT guidance subretinal injection to induce and multimodality imaging system to monitor CNV over time in rabbit eyes. The significance of our work lies in providing the optimal setting and conditions to make use of the OCT image guided system to improve the consistency and reproducibility of experimental results in subretinal injection induced CNV model in rabbits. For the first time, this study successfully demonstrated the dual-modality PAM-OCT system, without using exogenous contrast agents, can detect and visualize CNV in the rabbit eye with high resolution. This is promising system for diagnosing and monitoring CNV.

Molecular and cellular imaging of the eye.

The application of molecular and cellular imaging in ophthalmology has numerous benefits. It can enable the early detection and diagnosis of ocular diseases, facilitating timely intervention and improved patient outcomes. Molecular imaging techniques can help identify disease biomarkers, monitor disease progression, and evaluate treatment responses. Furthermore, these techniques allow researchers to gain insights into the pathogenesis of ocular diseases and develop novel therapeutic strategies. Molecular and cellular imaging can also allow basic research to elucidate the normal physiological processes occurring within the eye, such as cell signaling, tissue remodeling, and immune responses. By providing detailed visualization at the molecular and cellular level, these imaging techniques contribute to a comprehensive understanding of ocular biology. Current clinically available imaging often relies on confocal microscopy, multi-photon microscopy, PET (positron emission tomography) or SPECT (single-photon emission computed tomography) techniques, optical coherence tomography (OCT), and fluorescence imaging. Preclinical research focuses on the identification of novel molecular targets for various diseases. The aim is to discover specific biomarkers or molecular pathways associated with diseases, allowing for targeted imaging and precise disease characterization. In parallel, efforts are being made to develop sophisticated and multifunctional contrast agents that can selectively bind to these identified molecular targets. These contrast agents can enhance the imaging signal and improve the sensitivity and specificity of molecular imaging by carrying various imaging labels, including radionuclides for PET or SPECT, fluorescent dyes for optical imaging, or nanoparticles for multimodal imaging. Furthermore, advancements in technology and instrumentation are being pursued to enable multimodality molecular imaging. Integrating different imaging modalities, such as PET/MRI (magnetic resonance imaging) or PET/CT (computed tomography), allows for the complementary strengths of each modality to be combined, providing comprehensive molecular and anatomical information in a single examination. Recently, photoacoustic microscopy (PAM) has been explored as a novel imaging technology for visualization of different retinal diseases. PAM is a non-invasive, non-ionizing radiation, and hybrid imaging modality that combines the optical excitation of contrast agents with ultrasound detection. It offers a unique approach to imaging by providing both anatomical and functional information. Its ability to utilize molecularly targeted contrast agents holds great promise for molecular imaging applications in ophthalmology. In this review, we will summarize the application of multimodality molecular imaging for tracking chorioretinal angiogenesis along with the migration of stem cells after subretinal transplantation in vivo.

Advanced nanomaterials for imaging of eye diseases.

Background and purpose: Vision impairment and blindness present significant global challenges, with common causes including age-related macular degeneration, diabetes, retinitis pigmentosa, and glaucoma. Advanced imaging tools, such as optical coherence tomography, fundus photography, photoacoustic microscopy, and fluorescence imaging, play a crucial role in improving therapeutic interventions and diagnostic methods. Contrast agents are often employed with these tools to enhance image clarity and signal detection. This review aims to explore the commonly used contrast agents in ocular disease imaging. Experimental approach: The first section of the review delves into advanced ophthalmic imaging techniques, outlining their importance in addressing vision-related issues. The emphasis is on the efficacy of therapeutic interventions and diagnostic methods, establishing a foundation for the subsequent exploration of contrast agents. Key results: This review focuses on the role of contrast agents, with a specific emphasis on gold nanoparticles, particularly gold nanorods. The discussion highlights how these contrast agents optimize imaging in ocular disease diagnosis and monitoring, emphasizing their unique properties that enhance signal detection and imaging precision. Conclusion: The final section, we explores both organic and inorganic contrast agents and their applications in specific conditions such as choroidal neovascularization, retinal neovascularization, and stem cell tracking. The review concludes by addressing the limitations of current contrast agent usage and discussing potential future clinical applications. This comprehensive exploration contributes to advancing our understanding of contrast agents in ocular disease imaging and sets the stage for further research and development in the field.

Selective nanosecond laser removal of retinal pigment epithelium for cell therapy.

Retinal pigment epithelial (RPE) cells play a crucial role in the health of the retina, and their dysfunction is associated with various ocular diseases. The transplantation of RPE cells has been proposed as a potential treatment for numerous degenerative diseases, including geographic atrophy from macular degeneration. However, current models to induce RPE damage in animal models prior to transplantation involve mechanical scraping, chemical administration, or laser photocoagulation techniques, which can damage the overlying neurosensory retina. This study aims to investigate the feasibility and efficacy of nanosecond duration laser treatment to safely remove large areas of RPE cells without causing damage to the adjacent tissue or affecting the retinal architecture. Twelve pigmented rabbits were treated with a nanosecond laser on each eye at a laser energy ranging from 200 to 800 nJ with a treated area of 5 × 5 mm2. Human induced pluripotent stem cells-differentiated to RPE (hiPSC-RPE) cells labeled with indocyanine green (ICG), an FDA approved dye, were transplanted subretinally into the damaged RPE areas at day 14 post-laser treatment. The RPE atrophy and hiPSC-RPE cell survival was evaluated and monitored over a period of 14 days using color photography, fluorescein angiography (FA), photoacoustic microscopy (PAM), and optical coherence tomography (OCT) imaging. All treated eyes demonstrated focal RPE loss with a success rate of 100%. The injured RPE layers and the transplanted hiPSC-RPE cells were visualized in three dimensions using PAM and OCT. By performing PAM at an optical wavelength of 700 nm, the location of hiPSC-RPE cells were identified and distinguished from the surrounding RPE cells, and the induced PA signal increased up to 18 times. Immunohistochemistry results confirmed the grafted hiPSC-RPE replaced regions of RPE damage. This novel technique has the potential to serve as an animal model of RPE degeneration, to improve models of RPE transplantation, and may help accelerate translation of this therapeutic strategy for clinical use.

Multimodal Imaging-Guided Stem Cell Ocular Treatment.

Stem cell therapies are gaining traction as promising treatments for a variety of degenerative conditions. Both clinical and preclinical studies of regenerative medicine are hampered by the lack of technologies that can evaluate the migration and behavior of stem cells post-transplantation. This study proposes an innovative method to longitudinally image in vivo human-induced pluripotent stem cells differentiated to retinal pigment epithelium (hiPSC-RPE) cells by multimodal photoacoustic microscopy, optical coherence tomography, and fluorescence imaging powered by ultraminiature chain-like gold nanoparticle cluster (GNC) nanosensors. The GNC exhibits an optical absorption peak in the near-infrared regime, and the 7-8 nm size in diameter after disassembly enables renal excretion and improved safety as well as biocompatibility. In a clinically relevant rabbit model, GNC-labeled hiPSC-RPE cells migrated to RPE degeneration areas and regenerated damaged tissues. The hiPSC-RPE cells' distribution and migration were noninvasively, longitudinally monitored for 6 months with exceptional sensitivity and spatial resolution. This advanced platform for cellular imaging has the potential to enhance regenerative cell-based therapies.

Multimodal imaging of laser-induced choroidal neovascularization in pigmented rabbits.

This study aimed to demonstrate longitudinal multimodal imaging of laser photocoagulation-induced choroidal neovascularization (CNV) in pigmented rabbits. Six Dutch Belted pigmented rabbits were treated with 12 laser lesions in each eye at a power of 300 mW with an aerial diameter spot size of 500 µm and pulse duration of 100 ms. CNV progression was monitored over a period of 4 months using different imaging techniques including color fundus photography, fluorescein angiography (FA), photoacoustic microscopy (PAM), and optical coherence tomography (OCT). All treated eyes developed CNV with a success rate of 100%. The margin and morphology of CNV were detected and rendered in three dimensions using PAM and OCT. The CNV was further distinguished from the surrounding melanin and choroidal vessels using FDA-approved indocyanine green dye-enhanced PAM imaging. By obtaining PAM at 700 nm, the location and density of CNV were identified, and the induced PA signal increased up to 59 times. Immunohistochemistry with smooth muscle alpha-actin (αSMA) antibody confirmed the development of CNV. Laser photocoagulation demonstrates a great method to create CNV in pigmented rabbits. The CNV was stable for up to 4 months, and the CNV area was measured from FA images similar to PAM and OCT results. In addition, this study demonstrates that contrast agent-enhanced PAM imaging allows for precise visualization and evaluation of the formation of new blood vessels in a clinically-relevant animal model of CNV. This laser-induced CNV model can provide a unique technique for longitudinal studies of CNV pathogenesis that can be imaged with multimodal imaging.

Choroidal neovascularization removal with photo-mediated ultrasound therapy.

BACKGROUND: Age-related macular degeneration (AMD) is a major cause of irreversible central vision loss. The main reason for lost vision due to AMD is choroidal neovascularization (CNV). In the clinic, current treatments for CNV include photodynamic therapy, laser photocoagulation, and anti-vascular endothelial growth factor (VEGF) therapy. PURPOSE: This study evaluates a novel treatment technique combining synchronized nanosecond laser pulses and ultrasound bursts, namely photo-mediated ultrasound therapy (PUT) as a potential treatment method for CNV, for its efficacy and safety in the treatment of CNV via the experiments in a clinically-relevant rabbit model in vivo. METHODS: CNV was created by subretinal injection of Matrigel and vascular endothelial growth factor (M&V) in 10 New Zealand white rabbits. Six rabbits were used in the PUT group. In the control groups, two rabbits were treated by laser-only, and two rabbits were treated by ultrasound-only. The treatment efficacy was evaluated through fundus photography and fluorescein angiography (FA) longitudinally for up to 4 weeks. Rabbits were sacrificed for histopathology 3 months after treatment to examine the safety of PUT. RESULTS: The fluorescein leakage on FA was quantified to longitudinally evaluate treatment outcome. Compared with baseline, the relative intensity index was reduced by 26.57% ± 8.66% at 3 days after treatment, 27.24% ± 6.21% at 1 week after treatment, 27.79% ± 2.61% at 2 weeks after treatment, and 32.12% ± 3.23% at 4 weeks after treatment, all with a statistically significant difference of p < 0.01. The comparison between the relative intensity indexes from the two control groups (laser-only treatment and ultrasound-only treatment) did not show any statistically significant difference at all time points. Safety evaluation at 3 months with histopathology demonstrated that the PUT did not result in morphologic changes to the neurosensory retina. CONCLUSIONS: This study introduces PUT for the first time for the treatment of CNV. The results demonstrated good efficacy and safety of PUT to treat CNV in a clinically-relevant rabbit model. With a single session of treatment, PUT can safely reduce the leakage of CNV for at least 1 month after treatment.

Multimodal photoacoustic microscopy, optical coherence tomography, and fluorescence imaging of USH2A knockout rabbits.

Usher syndrome type 2A (USH2A) is a genetic disorder characterized by retinal degeneration and hearing loss. To better understand the pathogenesis and progression of this syndrome, animal models such as USH2A knockout (USH2AKO) rabbits have been developed. In this study, we employed multimodal imaging techniques, including photoacoustic microscopy (PAM), optical coherence tomography (OCT), fundus autofluorescence (FAF), fluorescein angiography (FA), and indocyanine green angiography (ICGA) imaging to evaluate the retinal changes in the USH2AKO rabbit model. Twelve New Zealand White rabbits including USH2AKO and wild type (WT) were used for the experiments. Multimodal imaging was implemented at different time points over a period of 12 months to visualize the progression of retinal changes in USH2AKO rabbits. The results demonstrate that ellipsoid zone (EZ) disruption and degeneration, key features of Usher syndrome, began at the age of 4 months old and persisted up to 12 months. The EZ degeneration areas were clearly observed on the FAF and OCT images. The FAF images revealed retinal pigment epithelium (RPE) degeneration, confirming the presence of the disease phenotype in the USH2AKO rabbits. In addition, PAM images provided high-resolution and high image contrast of the optic nerve and the retinal microvasculature, including retinal vessels, choroidal vessels, and capillaries in three-dimensions. The quantification of EZ fluorescent intensity using FAF and EZ thickness using OCT provided comprehensive quantitative data on the progression of degenerative changes over time. This multimodal imaging approach allowed for a comprehensive and non-invasive assessment of retinal structure, microvasculature, and degenerative changes in the USH2AKO rabbit model. The combination of PAM, OCT, and fluorescent imaging facilitated longitudinal monitoring of disease progression and provided valuable insights into the pathophysiology of USH2A syndrome. These findings contribute to the understanding of USH2A syndrome and may have implications for the development of diagnostic and therapeutic strategies for affected individuals. The multimodal imaging techniques employed in this study offer a promising platform for preclinical evaluation of potential treatments and may pave the way for future clinical applications in patients with Usher syndrome.

Renally Clearable Ultraminiature Chain-Like Gold Nanoparticle Clusters for Multimodal Molecular Imaging of Choroidal Neovascularization.

Currently, available gold nanoparticles (GNPs) typically accumulate in the liver and spleen, leading to concerns for their long-term biosafety. To address this long-standing problem, ultraminiature chain-like gold nanoparticle clusters (GNCs) are developed. Via self-assembly of 7-8 nm GNP monomers, GNCs provide redshifted optical absorption and scattering contrast in the near-infrared window. After disassembly, GNCs turn back to GNPs with a size smaller than the renal glomerular filtration size cutoff, allowing their excretion via urine. A one-month longitudinal study in a rabbit eye model demonstrates that GNCs facilitate multimodal molecular imaging of choroidal neovascularization (CNV) in vivo, non-invasively, with excellent sensitivity and spatial resolution. GNCs targeting αv ß3  integrins enhance photoacoustic and optical coherence tomography (OCT) signals from CNV by 25.3-fold and 150%, respectively. With excellent biosafety and biocompatibility demonstrated, GNCs render a first-of-its-kind nanoplatform for biomedical imaging.

USH2A Gene Mutations in Rabbits Lead to Progressive Retinal Degeneration and Hearing Loss.

Purpose: Mutations in USH2A gene are responsible for the greatest proportion of the Usher Syndrome (USH) population, among which more than 30% are frameshift mutations on exon 13. A clinically relevant animal model has been absent for USH2A-related vision loss. Here we sought to establish a rabbit model carrying USH2A frameshift mutation on exon 12 (human exon 13 equivalent). Methods: CRISPR/Cas9 reagents targeting the rabbit USH2A exon 12 were delivered into rabbit embryos to produce an USH2A mutant rabbit line. The USH2A knockout animals were subjected to a series of functional and morphological analyses, including acoustic auditory brainstem responses, electroretinography, optical coherence tomography, fundus photography, fundus autofluorescence, histology, and immunohistochemistry. Results: The USH2A mutant rabbits exhibit hyper-autofluorescent signals on fundus autofluorescence and hyper-reflective signals on optical coherence tomography images as early as 4 months of age, which indicate retinal pigment epithelium damage. Auditory brainstem response measurement in these rabbits showed moderate to severe hearing loss. Electroretinography signals of both rod and cone function were decreased in the USH2A mutant rabbits starting from 7 months of age and further decreased at 15 to 22 months of age, indicating progressive photoreceptor degeneration, which is confirmed by histopathological examination. Conclusions: Disruption of USH2A gene in rabbits is sufficient to induce hearing loss and progressive photoreceptor degeneration, mimicking the USH2A clinical disease. Translational Relevance: To our knowledge, this study presents the first mammalian model of USH2 showing the phenotype of retinitis pigmentosa. This study supports the use of rabbits as a clinically relevant large animal model to understand the pathogenesis and to develop novel therapeutics for Usher syndrome.

Multimodal In Vivo Imaging of Retinal and Choroidal Vascular Occlusion.

Photoacoustic microscopy (PAM) is an emerging retinal imaging technique that can provide high spatial resolution and high contrast of chorioretinal vessels. PAM is compatible with optical coherence tomography (OCT) and fluorescence imaging, allowing for development of a multimodal imaging system that combines these imaging modalities into one. This study presents a non-invasive, label-free in vivo imaging of retinal and choroidal vascular occlusion using multimodal imaging system, including PAM and OCT. Both retinal vein occlusion (RVO) and choroidal vascular occlusion (CVO) were clearly identified selectively using a spectroscopic PAM imaging. RVO and CVO were created in six rabbits using laser photocoagulation. The dynamic changes of retinal vasculature were observed and evaluated using color fundus photography, fluorescein angiography, OCT, and PAM. The position of RVO and CVO were imaged with different wavelengths ranging from 532 to 600 nm. The data shows that occluded vessels were clearly distinguished from the surrounding retinal vessels on the PAM images. This advanced imaging system is a promising technique for imaging retinal ischemia in preclinical disease models.

Chorioretinal Hypoxia Detection Using Lipid-Polymer Hybrid Organic Room-Temperature Phosphorescent Nanoparticles.

Ischemia-induced hypoxia is a common complication associated with numerous diseases and is the most important prognostic factor in retinal vein occlusions (RVOs). Early detection and long-term visualization of retinal tissue hypoxia is essential to understand the pathophysiology and treatment of ischemic retinopathies. However, no effective solution exists to evaluate extravascular retinal tissue oxygen tension. Here, we demonstrate a lipid-polymer hybrid organic room-temperature phosphorescence (RTP) nanoparticle (NP) platform that optically detects tissue hypoxia in real-time with high signal-to-noise ratio. The fabricated NPs exhibit long-lived bright RTP, high sensitivity toward oxygen quenching, and desirable colloidal and optical stability. When tested as a hypoxia imaging probe in vivo using rabbit RVO and choroidal vascular occlusion (CVO) models via intravitreal and intravenous (IV) injections, respectively, its RTP signal is exclusively turned on where tissue hypoxia is present with a signal-to-noise ratio of 12.5. The RTP NP platform is compatible with multimodal imaging. No ocular or systemic complications are observed with either administration route. The developed organic RTP NPs present a novel platform approach that allows for biocompatible, nondestructive detection of tissue hypoxia and holds promise as a sensitive imaging tool to monitor longitudinal tissue oxygen levels and evaluate various hypoxia-driven vascular diseases.

Biodegradable silicon nanoneedles for ocular drug delivery.

Ocular drug delivery remains a grand challenge due to the complex structure of the eye. Here, we introduce a unique platform of ocular drug delivery through the integration of silicon nanoneedles with a tear-soluble contact lens. The silicon nanoneedles can penetrate into the cornea in a minimally invasive manner and then undergo gradual degradation over the course of months, enabling painless and long-term sustained delivery of ocular drugs. The tear-soluble contact lens can fit a variety of corneal sizes and then quickly dissolve in tear fluid within a minute, enabling an initial burst release of anti-inflammatory drugs. We demonstrated the utility of this platform in effectively treating a chronic ocular disease, such as corneal neovascularization, in a rabbit model without showing a notable side effect over current standard therapies. This platform could also be useful in treating other chronic ocular diseases.

A lipid nanoparticle platform for mRNA delivery through repurposing of cationic amphiphilic drugs.

Since the recent clinical approval of siRNA-based drugs and COVID-19 mRNA vaccines, the potential of RNA therapeutics for patient healthcare has become widely accepted. Lipid nanoparticles (LNPs) are currently the most advanced nanocarriers for RNA packaging and delivery. Nevertheless, the intracellular delivery efficiency of state-of-the-art LNPs remains relatively low and safety and immunogenicity concerns with synthetic lipid components persist, altogether rationalizing the exploration of alternative LNP compositions. In addition, there is an interest in exploiting LNP technology for simultaneous encapsulation of small molecule drugs and RNA in a single nanocarrier. Here, we describe how well-known tricyclic cationic amphiphilic drugs (CADs) can be repurposed as both structural and functional components of lipid-based NPs for mRNA formulation, further referred to as CADosomes. We demonstrate that selected CADs, such as tricyclic antidepressants and antihistamines, self-assemble with the widely-used helper lipid DOPE to form cationic lipid vesicles for subsequent mRNA complexation and delivery, without the need for prior lipophilic derivatization. Selected CADosomes enabled efficient mRNA delivery in various in vitro cell models, including easy-to-transfect cancer cells (e.g. human cervical carcinoma HeLa cell line) as well as hard-to-transfect primary cells (e.g. primary bovine corneal epithelial cells), outperforming commercially available cationic liposomes and state-of-the-art LNPs. In addition, using the antidepressant nortriptyline as a model compound, we show that CADs can maintain their pharmacological activity upon CADosome incorporation. Furthermore, in vivo proof-of-concept was obtained, demonstrating CADosome-mediated mRNA delivery in the corneal epithelial cells of rabbit eyes, which could pave the way for future applications in ophthalmology. Based on our results, the co-formulation of CADs, helper lipids and mRNA into lipid-based nanocarriers is proposed as a versatile and straightforward approach for the rational development of drug combination therapies.

Laser-induced nanobubbles safely ablate vitreous opacities in vivo.

In myopia, diabetes and ageing, fibrous vitreous liquefaction and degeneration is associated with the formation of opacities inside the vitreous body that cast shadows on the retina, appearing as 'floaters' to the patient. Vitreous opacities degrade contrast sensitivity function and can cause notable impairment in vision-related quality of life. Here we introduce 'nanobubble ablation' for safe destruction of vitreous opacities. Following intravitreal injection, hyaluronic acid-coated gold nanoparticles and indocyanine green, which is widely used as a dye in vitreoretinal surgery, spontaneously accumulate on collagenous vitreous opacities in the eyes of rabbits. Applying nanosecond laser pulses generates vapour nanobubbles that mechanically destroy the opacities in rabbit eyes and in patient specimens. Nanobubble ablation might offer a safe and efficient treatment to millions of patients suffering from debilitating vitreous opacities and paves the way for a highly safe use of pulsed lasers in the posterior segment of the eye.

Functionalized contrast agents for multimodality photoacoustic microscopy, optical coherence tomography, and fluorescence microscopy molecular retinal imaging.

Near-infrared (NIR) targeting contrast agents have been investigated as great photoabsorbers to improve photoacoustic microscopy (PAM), OCT, and fluorescence imaging contrast for visualization of various diseases. In ophthalmology, a limited number of NIR contrast agents have been approved for clinical use. Recently, gold nanoparticles with different size and shapes have been developed for molecular imaging. This chapter provides the principles of multimodality PAM, OCT, and fluorescence imaging as well as a brief overview of contrast agents for optical imaging. A detailed protocol for the fabrication of discrete colloidal gold nanoparticles (GNPs), synthesis of functionalized RGD-conjugated chain-like GNP (CGNP) clusters labeled with indocyanine green (ICG) fluorescence dye (ICG@CGNP clusters-RGD), and validation of the synthesized nanoparticles to evaluate newly developed blood vessels in the retina, named choroidal neovascularization (CNV), is described. Using RGD peptide, ICG@CGNPs clusters-RGD can bind integrin which is expressed on activated endothelial cells and newly developed CNV. The targeting efficiency of nanoparticles is monitored by multimodality PAM, OCT, and fluorescence imaging longitudinally.