Near-Infrared Activated Liposomes for Neuroprotection in Glaucoma

Introduction: Glaucoma, a serious eye condition, leads to optic nerve degeneration and potential blindness. Current treatments focus on reducing intraocular pressure, the main modifiable risk factor, through pharmacological interventions, laser therapy, and surgery. However, these methods have limitations, including poor patient compliance, invasive procedures, and potential side effects.

To address these issues, an on-demand drug release mechanism triggered by non-invasive stimuli has been developed. This approach ensures localized, sustained drug delivery to retinal tissues. Near-infrared (NIR) stimuli, benefiting from deep tissue penetration and rapid response time, are used due to their compatibility with the eye’s natural processes. The localized surface plasmon resonance effect (LSPR) of nanomaterials is crucial in this context. Upon light illumination, the surface electrons of metallic nanoparticles oscillate, enhancing the electromagnetic field and leading to increased light absorption and scattering. By integrating LSPR with thermally responsive liposomal nanocarriers, a remotely controlled mechanism for on-demand local drug release is achieved.
Building on previous advancements, we developed an NIR-triggered drug release system using AuNRs and the hormone melatonin, known for its potential to alleviate retinal ganglion cell degeneration. Melatonin is encapsulated within liposomal carriers using 2-Hydroxypropyl-β-cyclodextrin (HPβCD) to enhance solubility and loading efficiency. A single injection followed by NIR illumination initiates drug release. In an acute glaucoma model, we observed significant reductions in intraocular pressure, improved retinal ganglion cell preservation, and reduced glial activation. This proof-of-concept application suggests our NIR-triggered drug delivery system as a promising therapeutic option for glaucoma treatment.

Materials and

Methods: Photothermal liposomes were synthesized using AuNRs, HP-β-CD, and melatonin. The lipids were dissolved in dichloromethane, mixed with AuNRs and HP-ꞵ-CD-melatonin complex, and subjected to vortexing at 3,000 rpm and sonication. The resultant emulsion underwent vacuum evaporation for 30 minutes and centrifugation at 10,000 rpm for 7 minutes for purification. Particle size was assessed using Nanoparticle Tracking Analysis. Thermal release studies were conducted on liposomes encapsulating Calcein, with fluorescence readings captured at 5-minute intervals. Biocompatibility tests were performed with HEK293FT cells, supplemented with 1 µL of liposome/DMSO suspensions for 24 hours. Cell viability was assessed using Calcein-AM and Ethidium Homodimer-1. The photothermal properties of AuNRs were validated using a temperature measurement assay, recording real-time temperature changes over 100 seconds post-NIR laser irradiation. Melatonin/Oil Red O and HP-β-CD inclusion complex was synthesized using microwave-assisted techniques, subjected to microwave irradiation at 700 W for 60 seconds. C57BL/6J mice were intravitreally injected with liposomes, followed by oil beads injection in the anterior chamber for intraocular pressure assessments. The mice underwent treatment using an 808 nm NIR Laser every three days over a 14-day period. For immunohistochemistry, the animals were euthanized and perfused with phosphate-buffered saline and 4% paraformaldehyde to fix the tissues. The retinal tissues were dissected, post-fixed, and cryoprotected. Thereafter, the tissues were washed, blocked, and incubated with primary and secondary antibodies. The retinas were then prepared for microscopy and quantitative analyses were conducted using Image J software.

Results, Conclusions, and Discussions: Encapsulated AuNRs in liposomes elevate local temperature under NIR light, enhancing melatonin diffusion. A 50 µL AuNRs solution, irradiated with an 11.2 mW NIR laser for 100 seconds, experienced a 4°C temperature rise. Liposomes, with a predefined lipid ratio, increased in mean diameter from 121.7 nm to 141.4 nm post-AuNRs encapsulation. Release kinetics from thermosensitive liposomes, assessed using calcein dye, displayed temperature sensitivity and a direct correlation between temperature and fluorescence signal. NIR-triggered release demonstrated a gradual increase in fluorescence intensity with exposure time, indicating time-dependent release from the liposomes upon NIR stimulation. HPβCD enhances melatonin's solubility by forming inclusion complexes. Cytotoxicity assessment of AuNRs liposomes on HEK293 cultures showed no significant variance in cell death rate, indicating biocompatibility. Immunohistochemical assessment after in vivo injection showed no significant changes in RGC numbers, GFAP expression levels, or GFAP distribution depths between the control group and those receiving AuNRs liposomes and NIR treatments, indicating safety and biocompatibility. In an acute glaucoma mouse model, NIR treatment significantly reduced IOP by (24±7) % compared to controls after 15 days. Immunohistochemical analysis showed reduced GFAP depth by (75±6) % and increased RGC count by (77±6) % in the group receiving AuNRs liposomes and NIR treatment, indicating neuroprotection and reduced gliosis. This approach offers a non-invasive, site-specific, and efficient drug delivery method, potentially advancing vision disease therapy.

Acknowledgements (Optional): This study was supported by the Binghamton University Faculty start-up funding (Q.W. and S.R.), Binghamton University Small Scale Systems Integration and Packaging (S3IP) Center of Excellence, funded by New York Empire State Development’s Division of Science, Technology, and Innovation (Q.W. and S.R.). We also thank the core facilities, Animal Care Services, Dr. Md Mofizur Rahman, Benjamin Morrow, Kristine Rowe, Winny Guan Zheng, Julianna Ayres, Dr. Kaimin Ye and Dr. Yuan Wan for their assistance.