New nanoparticle-based delivery systems are being developed to prolong drug retention time, improve drug penetration across ocular barriers and ensure sustained drug release with reduced toxicity and improved efficacy (Masse et al., 2019a; Omerović and Vranić, 2020; Yang et al., 2023a, 2023b; Ger et al., 2025). However, it is important to understand the biodistribution profiles in the eye of each nanoparticle developed depending on the procedure of administration selected to determine optimal ophthalmic formulations. Biodistribution studies in the eye are difficult to reproduce, not only because of the many barriers that make up the eye, but also because of the route of administration used, which can influence the fate of nanoparticles as drug delivery systems (Agrahari et al., 2016; Willcox et al., 2017; Srinivasarao et al., 2019; Koppa Raghu et al., 2020; Swetledge et al., 2021). Currently, there is no standardized protocol for in vivo ocular biodistribution enabling the results observed to be compared.

Designing, interpreting and compiling research on the nanoparticles ocular biodistribution raise major challenges. (i) There are countless ways to design a nanoparticle-based ocular delivery system, and any modification of size, charge, surface ligands, release profile and route of administration can have a considerable impact on the biodistribution and performance in the eye (Swetledge et al., 2021; Li and Huang, 2008; Feliu et al., 2016; Sonntag et al., 2021). (ii) Various animal models have been and can be used to test ocular biodistribution (Agrahari et al., 2016; Sonntag et al., 2021; Apaolaza et al., 2020; Raîche-Marcoux et al., 2025). However, the choice of an animal model has a strong impact on the ocular biodistribution profiles of administered nanoparticles, due to variations in eye size and physiological anatomy across different species (Loiseau et al., 2023). (iii) Among the multiple ways to track nanoparticles in the eye, the in vivo spatiotemporal distribution of nanoparticles is generally measured by time points after animal euthanasia and eye collection (Agrahari et al., 2016; Swetledge et al., 2021; Sonntag et al., 2021; Apaolaza et al., 2020).

Of all ocular administration methods, eye drops account for 90 % of all ophthalmic treatments, even though drug bioavailability is low, with less than 0.02 % of therapeutic molecules reaching the anterior chamber (Masse et al., 2019a; Ouellette et al., 2018; Jumelle et al., 2020; Vaneev et al., 2021). Conventional drug formulations are generally characterized by low retention times in the tear film, insufficient contact with the corneal epithelium, rapid elimination and difficulties in crossing ocular tissue barriers (Agrahari et al., 2016; Srinivasarao et al., 2019; Ouellette et al., 2018; Vaneev et al., 2021). To tackle these challenges, an in vivo ocular biodistribution study was conducted on patented modified gold nanoparticles developed in our laboratory as drug delivery systems for topical application (Raîche-Marcoux et al., 2025; Ouellette et al., 2018; Masse et al., 2019b; Raiche-Marcoux et al., 2022). Gold nanoparticles are the most widely studied metallic nanoparticles for biomedical applications due to their ease of fabrication and surface modification, their excellent chemical stability and their biocompatibility (Masse et al., 2019a; Li et al., 2019; Balfourier et al., 2020; Chen et al., 2021; Luo et al., 2021; Ghosh et al., 2025). These nanomaterials can be reproducibly synthesized, easily functionalized on their surface and, in some cases, characterized with atomic precision. Recent literature has shown that gold nanoparticles have ocular drug encapsulation and delivery abilities, as well as ultrastable and mucoadhesive properties, making them promising candidates for drug delivery systems in ophthalmology (Masse et al., 2019a, 2019b; Raîche-Marcoux et al., 2025; Ouellette et al., 2018; Raiche-Marcoux et al., 2022).

The optimization of each step of the biodistribution study has led us to propose a standardized in vivo protocol, including eye drop application (that can generate considerable variability), anesthesia and euthanasia, enucleation, eye dissection into various ocular tissues and their digestion, as well as their ex vivo analysis of gold (Au) atom content from gold nanoparticles using inductively coupled plasma-mass spectrometry (ICP-MS). ICP-MS offers very interesting analytical capabilities such as multi-elemental and isotopic determinations such as Au content in a wide variety of biological matrices with excellent detection limits (in the ppt range) while minimizing matrix effects (Sonntag et al., 2021; Lores-Padín et al., 2021; Laradji et al., 2021; Millar et al., 2022). Due to the many physical and physiological barriers in the eye that limit the penetration of substances contained in eye drops (Masse et al., 2019a; Raîche-Marcoux et al., 2025; Loiseau et al., 2023), ICP-MS enables fine detection of Au content down to trace levels in various ocular tissues after administration of a gold nanoparticle suspension. In ophthalmology, rabbit proved to be a convenient alternative animal model, bridging the gap between rodents and non-human primates, as it shares many anatomical and physiological features with humans, including eye size, internal structures, and optical system, as well as conjunctival cavity volume (Loiseau et al., 2023). Among these features, the size of rabbit eyes facilitates drug administration and surgical procedures, enabling the collection of sufficient biological tissues either in vivo or post-mortem for quantitative analyses (Loiseau et al., 2023; Y Zernii et al., 2016). However, standardization of the in vivo protocol is necessary, as the limitations of this approach with larger animal models are both financial and ethically challenging from a scientific perspective. The accuracy of the biodistribution data obtained is thus limited by the frequency of time points, particularly with regard to Russell and Burch's Three Rs principle (replacement, reduction and refinement), which is a useful concept for the scientific and ethical evaluation of the use of animals in scientific studies (Clark, 2018), limiting the number of animals used, since in our case the animal often has to be sacrificed before the eyes are collected. Researchers need to determine whether animals are necessary or if alternatives are available to replace them. If animals are required, researchers must determine the number required to obtain valid results while maximizing the amount of data obtained from each animal. They must also identify possible sources of suffering and ways to reduce them. The Three Rs principle is part of any animal experimental design.

This technical report describes all the experimental procedures relating to the biodistribution of gold nanoparticles for any selected exposure time and successful reproducibility in another laboratory to overcome the various challenges such as reproducibility of eye drop deposition, speed of medical and surgical procedures in rabbits, eye dissection with a fast, precise and pure manner as well as digestion steps of these ocular tissues, while minimizing handling and avoiding cross-contamination. Finally, we have detailed the entire ICP-MS analysis procedure, as to our knowledge, few facilities have the expertise to measure gold (Au) quantities using this analytical technique. This would avoid memory effects due to the device, as well as any interference with the biological matrix or other contamination during gold atom analysis.

Although we describe a detailed in vivo protocol for eye drop administration, the procedures for enucleation, eye dissection and digestion as well as ex vivo analysis of gold content by ICP-MS can also be applied to other routes of ocular administration. The standardization of in vivo protocols in ocular biodistribution studies of gold-based delivery nanosystems could meet a need expressed by all ophthalmic surgeons to improve eye treatments, i.e., (i) reduce the frequency of administration during treatments, and (ii) increase the drug residence time to improve the therapeutic efficacy.