## 2 Discussion
### Limitations, Safety and Challenges
While all the aforementioned studies did not exhibit toxic effects on the CECs, for up to 4 \(\upmu\)m-sized microparticles, it is vital to consider long-term implications of utilising MNPs. In vitro cells cultured with nanoparticles for longer periods of time (30 days) have been shown to have significantly lower amounts of intracellular nanoparticles, suggesting that after being endocytosed, nanoparticles may be removed from the cell by exocytosis or lysosomal degradation [80; 98]. If an equivalent process takes place in vivo, it may permit MNPs to be dissociated from the transplanted HCECs, and either affect trabecular meshwork function, or exhibit intraocular or systemic toxicity. Another critical safety consideration regarding the use of MNPs, particularly iron-based ones in ocular applications, pertains to magnetic hyperthermia. This phenomenon involves MNPs acting as nano heaters, inducing localised heat in ocular tissues when exposed to an alternating magnetic field [99]. This is of particular concern due to the high sensitivity and vulnerability of ocular tissues to heat and rapid temperature fluctuations [100]. Furthermore, MNPs activated during magnetic hyperthermia can generate ROS, leading to DNA and mitochondrial damage, as well as protein oxidation [99]. In magnetic hyperthermia, the oscillation of MNPs in response to an alternating magnetic field converts absorbed energy into heat within targeted tissues, such as tumours. While this therapeutic approach has demonstrated efficacy in treating cancers like retinoblastoma, with minimal impact on non-tumour cells, its application in ocular settings necessitates careful consideration due to the eye's sensitivity [101]. Importantly, the reviewed studies did not explicitly discuss magnetic hyperthermia as an adverse effect. Therefore, proactive measures are essential to anticipate and mitigate potential risks. These include precise monitoring and characterisation of MNP distribution and concentration, as well as meticulous control of magnetic field application to avoid unintended heat generation and mitigate the risk of tissue damage. Immunogenicity concerns related to MNPs derived from animal proteins highlight potential risks in human applications, necessitating thorough evaluation to ensure compatibility and safety. In certain investigations, animal immunoglobulins were utilised, as illustrated in the protocol by [PERSON] et al., which employed rat anti-mouse IgG1 superparamagnetic MACS MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany). Notably, no immunogenic reactions were observed in these studies [80; 81]. However, the transfer of non-human-derived protein to human subjects raises concerns about increased risk of immunogenic reaction to the HCECs. Although the aforementioned studies noted that ROS production following MNP cellular uptake did not affect cell viability in the corneal endothelium, it is crucial to consider the potential impact of ROS generation on nearby sensitive tissues, such as the lens and retina [102]. Research indicates that exposure of retinal cells to silver and gold nanoparticleselevates ROS levels, thereby triggering microglial activation within retinal tissue [103; 104]. [PERSON] et al. demonstrated that exposure to 80 nm silver nanoparticles resulted in a significant 30% increase in activated microglia, while exposure to 20 nm and 80 nm gold nanoparticles led to increases of 15% and 11% in activated microglia, respectively [103]. This phenomenon is of particular concern due to its association with photoreceptor apoptosis and the progression to retinal degeneration [104]. Within the lens, cerium oxide nanoparticles (nanoceria) demonstrate antioxidative properties that alleviate oxidative stress and inhibit protein glycation in human lens epithelial cells (HLECs) [105]. Nanoceria effectively permeate HLECs, thereby enhancing the reduced to oxidised glutathione ratio (GSH/GSSG) and attenuating glucose-induced protein glycation, both pivotal mechanisms in cataract formation [105].