Gemcitabine (2′,2′-difluoro-2′-deoxycytidine) (Gem) is a nucleoside analog of 2′-deoxycytidine with a major clinical interest, approved against a wide variety of solid tumors (Toschi et al., 2005). Although highly effective, Gem is associated with notable side effects, mainly hematological (anemia, neutropenia, thrombocytopenia) and gastro-intestinal (nausea and vomiting) (Toschi et al., 2005). In addition, high doses are often required because of the rapid blood clearance of the drug (elimination half-life of 8 min), mainly due to deamination in inactive 2′,2′-difluorodeoxyuridine (Abbruzzese et al., 1991). Besides, for activation, the drug first needs to be transported through human nucleoside transporters (hNT), before being converted to gemcitabine-triphosphate (GemTP), its active form (Mackey et al., 1999, Plunkett et al., 1995).

To address these issues, researchers have explored encapsulating Gem in “stealth” (prolonged blood circulation ability) nanocarriers. Two main strategies have been pursued: i) encapsulation of the single agent Gem, for instance in albumin or silica nanoparticles (Dubey et al., 2015, Saini et al., 2020) and ii) chemical modification of Gem with a lipophilic moiety to allow its encapsulation into liposomal or nanoparticle formulations (Arpicco et al., 2013, Bekkara-Aounallah et al., 2008, Chen et al., 2021, Stella et al., 2007). In the first case, poor drug payloads (up to 15 %) and limited encapsulation efficiencies (up to 57 %) were obtained (Saini et al., 2020). The second strategy involved delicate synthesis and depended on the cells’ ability to metabolize the prodrug effectively (Chen et al., 2021).

In this context, porous nanosized Metal Organic Frameworks (MOFs), and more particularly iron trimesate MIL-100(Fe) (MIL stands for Materials of Institute Lavoisier), allow the encapsulation of Gemcitabine-monophosphate (GemMP). This provides an additional advantage by circumventing the rate-limiting step involved in the drug’s activation, its first phosphorylation. The strong interaction between the phosphate moiety of GemMP, and the Fe3+ trimers in the structure of MIL-100(Fe) MOFs allowed to achieve unprecedented drug loadings (up to ∼ 30 wt%), with an almost perfect encapsulation efficiency (>98 %) (Rodriguez-Ruiz et al., 2015).

MIL-100(Fe) MOFs are porous nanoparticles made up of iron Fe3+ trimers and trimesate as the organic ligand. These materials possess the remarkable ability to be impregnated with drugs using environmentally friendly methods that do not involve organic solvents (Agostoni et al., 2013c, Horcajada et al., 2010, Horcajada et al., 2007, Rodriguez-Ruiz et al., 2015). The MIL-100(Fe) MOFs’ unique composition makes them particularly interesting for biomedical applications, as they have already been shown to decompose into biocompatible and poorly toxic components in vivo (Baati et al., 2013). However, MIL-100(Fe) MOFs were rapidly cleared after intravenous administration and tend to accumulate mainly in lungs and liver (Baati et al., 2013, Simon-Yarza et al., 2016). To ensure stealth properties, the MOFs therefore need to be coated (Ettlinger et al., 2022).

Among the various strategies for coating MIL-100(Fe) MOFs with hydrophilic shells, polymers containing phosphate moieties proved to be particularly effective, as they spontaneously coordinate with the iron sites available on the external surface (Agostoni et al., 2015, Li et al., 2020b, Wuttke et al., 2015). Initial research concentrated on cyclodextrin-phosphate derivatives, which spontaneously coated MOFs, achieving a significant coated amount of 17 wt% (Agostoni et al., 2015). More recently, the introduction of the dextran-alendronate-poly(ethylene glycol) (DAP) led to one of the highest coating levels, surpassing 30 wt% (Cutrone et al., 2019).

However, the current understanding of DAP-coated MIL-100(Fe) is limited in terms of ability to encapsulate cytotoxic drugs and the effectiveness of GemMP-loaded MOFs@DAP. Additionally, previous in vitro studies with uncoated GemMP loaded MIL-100(Fe) using monolayer cells (Cutrone et al., 2019, Li et al., 2020b, Rodriguez-Ruiz et al., 2015), lack the complexity of tissue penetration and do not account for the in vivo activity of the MOFs. In this study, we introduce the chorioallantoic membrane (CAM) model, which provides an alternative to mice as an in vivo model, particularly for screening of nanoparticle toxicity and drug activity on tumor growth inhibition.

In this model, human tumors can be easily established on the CAM, the membrane surrounding the embryos of fertilized avian eggs, by taking advantage of the poor immune system of chick embryos and the rich vascularization of the membrane. These tumors evolve with vascular features and the circulation system typical of the in vivo environment (Victorelli et al., 2020). Thanks to the vascularized nature of the CAM, the blood flow helps with nutrient transfer and waste removal, providing insights on drug clearance of treatments tested in this model.

The CAM can be easily accessed either by opening a window at the level of an air pocket (in ovo) or by extracting the live embryo and its tissues from the egg (ex ovo). This model attracts increased attention, as it allows for screening of a large number of treatment conditions in vivo, while refining the number of rodents and reducing their suffering. Indeed, the intervention takes place on the CAM, which lacks nerves instead of directly on the embryos (Kundeková et al., 2021). As a result, researchers focused on the in ovo studies of drug delivery systems (DDS) to gather more robust data, that align with the core principles of the 3Rs (Vargas et al., 2007, Vu et al., 2018).

Previous work showed the interest of using encapsulated Gem to improve the drug’s efficacy against castration-resistant prostate cancer (CRPC) (Jantscheff et al., 2009). In this study, we therefore chose to implement CRPC xenografts of DU145 in ovo to investigate the efficiency of surface modified GemMP-loaded MOFs.

In substance, this study aims to: i) optimize the DAP coating onto MOFs; ii) investigate the impact of DAP coating on the stability of MOFs and the encapsulation efficiency of GemMP; iii) decipher the role of the DAP coating in the MOFs’ interaction with macrophages and DU145 prostate cancer cells; and iv) use the in ovo CAM assay to showcase the potential of encapsulating GemMP within DAP-coated MOFs.