Abstract
Purpose :
Current treatment of endothelial diseases (e.g. Fuch’s dystrophy) is limited to corneal transplantation which is hampered by the availability of sufficient qualitative grafts, limiting the number of patients that can be treated. New pharmacological alternatives, e.g. nucleic acids (NA) inhibiting the Rho kinase pathway, which could stimulate the proliferation of corneal endothelial cells (CEC), are of current interest. However, selectively delivering NA to CEC is challenging due to several corneal barriers and lysosomal degradation of NA. Using in vitro and ex vivo models, we investigated if intracameral injection of NA combined with photoporation, a technique creating transient pores in cell membranes by imploding vapor nanobubbles (VNB’s) originating from pulsed laser light irradiation of photosensitizers, overcomes these barriers. Additionally, embedding NA with photosensitizers, i.e. gold nanoparticles (AUNP), in a ready-to-use hyaluronic acid (HA) gel, commonly used to protect CEC during cataract surgery, reduces off-target toxicity and allows AUNP removal after photoporation.
Methods :
Immortalized human CEC (HCEC-B4G12), bovine corneas and human corneal explants were used as in vitro and ex vivo models for delivery of FITC-dextran (FD) 150, FD 500 and eGFP mRNA to CEC. Two AUNP sizes, 60 nm and 100 nm, were irradiated by a 561 nm pulsed laser (<7ns; 1.05 J/ cm2 for 60 nm and 1.6 J/cm2 for 100 nm AUNP) to create VNB’s. These particles were also embedded in a HA (1000-2000 kDa) gel. Optimization was performed in vitro by investigating transfection efficiency and viability by flow cytometry and CellTiterGLo assay respectively. Microscopy was used to detect ex vivo transfection on both models with the optimized conditions.
Results :
Photoporation using 60 nm AUNP was able to deliver FD150 as a free suspension or in a HA gel, but was unable to deliver FD500 and eGFP mRNA. Using 100 nm AUNP resulted in successful delivery of FD500 both as suspension and inside HA gel. Delivery of eGFP mRNA in vitro resulted in 26,74% (+/- 2.83%) of cells expressing eGFP, measured 24h after treatment. Delivery of eGFP mRNA was also successful in ex vivo human CEC.
Conclusions :
Photoporation was able to deliver FD 150 with 60 nm AUNP while 100 nm AUNP successfully delivered FD500 and eGFP mRNA to human CEC. Embedding AUNP in a ready-to-use HA gel also resulted in successful in vitro and ex vivo eGFP mRNA delivery.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.