Quantitative comparison and toxicological evaluation of DNA-based nanoparticles for the treatment of anterior eye segment diseases

Abstract

Purpose: Last year we presented an example of a new class of DNA-based nanoparticles (NPs) to overcome current shortcomings in the treatment of anterior eye segment diseases. Here, we will present a variety of DNA-based NPs exhibiting different affinity to the eye surface. By tailoring the duration time of the medication a lower regime can be attained, resulting in an improved compliance. In addition, it allows for a lower drug concentration, what can lead to fewer side effects.

Methods: To evaluate the NPs, they were conjugated to a fluorescent marker and dropped on pig eyes (ex-vivo) and rat eyes (conscious animals). Incubation time was 5, 15 and 30 min, and 30 min, 2h and 24h, respectively. Additionally, for the best NP the maximum adherence time was investigated. After sacrificing the animal the eyes were counterstained and imaged by fluorescence microscopy. Additional assessment of the adhesion of the NPs was performed using an ocular fluorophotometer and antibody staining. The toxicity of the NPs was tested on three different ocular cell lines including cell viability and apoptosis assays. Additionally, in-vivo safety was evaluated via TUNEL staining. Further tests were performed with different buffers for the NPs.

Results: In this study 8 different DNA-based NPs were tested in pig eyes. Of them 7 showed a good or excellent adhesion to the cornea. These were further tested on conscious rats with 3 different buffer solutions, where 6 showed good binding to the rat cornea epithelium for at least 30 min after application. Two hours after application 4 still showed positive results. The best carrier was even detectable on the cornea after 4 h. Antibody staining against Zo-1, Galectin 3 & Mucins allowed localization of the NPs. Regarding toxicity evaluation, our NP showed to be safe as no toxicity at all was found with the nanoparticle that showed best adhesion to the cornea.

Conclusions: In this study we found that by changing the properties of the NP, i.e. length and number of modified bases, we are able to influence the adhesion time of the NP to the cornea. Through this the exposure time of medication to the cornea can be optimized, allowing us to personalize our drug-carrier. Additionally, no toxic effects of the carrier were found. This important finding allows the safe utilization of the NP platform for ocular drug delivery.