Lactoferrin (Lf) is a globular glycoprotein found mainly in milk. It has a very high affinity for iron(III) ions, and its fully saturated form is called holoLf. The antimicrobial, antiviral, anticancer, and immunomodulatory properties of Lf have been studied extensively for the past two decades. However, to demonstrate therapeutic benefits, Lf has to be efficiently delivered to the intestinal tract in its structurally intact form. This work aimed to optimize the encapsulation of holoLf in a system based on the versatile Eudragit^® RS polymer to protect Lf against the proteolytic environment of the stomach. Microparticles (MPs) with entrapped holoLf were obtained with satisfactory entrapment efficiency (90–95%), high loading capacity (9.7%), and suitable morphology (spherical without cracks or pores). Detailed studies of the Lf release from the MPs under conditions that included simulated gastric or intestinal fluids, prepared according to the 10th edition of the European Pharmacopeia, showed that MPs partially protected holoLf against enzymatic digestion and ionic iron release. The preincubation of MPs loaded with holoLf under conditions simulating the stomach environment resulted in the release of 40% of Lf from the MPs. The protein released was saturated with iron ions at 33%, was structurally intact, and its iron scavenging properties were preserved.

The growth of Lactobacillus plantarum, a member of the Lactobacillus genus, which plays a crucial role in the bacterial microbiome of the gut, is significantly influenced by manganese ions. They can be safely delivered to the intestines by exploiting the chelating abilities of lactoferrin. The aim of this work was to encapsulate lactoferrin saturated with manganese ions (MnLf) in a system based on the Eudragit^® RS polymer to protect protein from degradation and manganese release in the gastric environment. The entrapment efficiency was satisfactory, reaching about 95%, and most importantly, manganese ions were not released during microparticles (MPs) formation. The release profile of the protein from the freshly prepared MPs was sustained, with less than 15% of the protein released within the first hour. To achieve similar protein release efficiency, freeze-drying was carried out in the presence of 10% (w/v) mannitol as a cryoprotectant for MPs frozen at −20 °C. MPs with encapsulated MnLf exhibited prebiotic activity towards Lactobacillus plantarum. More importantly, the presence of equivalent levels of manganese ions in free form in the medium, as well as chelating by lactoferrin encapsulated in MPs, had a similar impact on stimulating bacterial growth. This indicates that the bioavailability of manganese ions in our prepared system is very good.

Hypoxia, defined as a reduction in oxygen availability resulting from an imbalance between oxygen supply and demand, represents an attractive tumor vulnerability that can be exploited to activate diagnostic and therapeutic agents. Despite the expected selectivity of hypoxiaactivated prodrugs (HAPs), clinical studies have demonstrated relatively low efficacy. This limitation is attributed to multiple factors, as hypoxiainduced cellular responses are highly complex and strongly patientdependent, highlighting the need for reliable hypoxia markers to enable appropriate patient stratification. In this study, we investigated the relationships between the expression levels of selected oxidoreductases involved in activation of HAPs, the readout of recently developed fluorescent hypoxia sensor, and the cytotoxicity of HAP - evofosfamide under rigorously controlled oxygen conditions (21% O₂ vs 1% O₂) across a panel of six human cancer cell lines (A375, A2058, MCF7, PANC1, A549, and HT29). Additionally, we assessed HIF1α expression and compared results obtained using three laboratory models of hypoxia: a hypoxic chamber and chemical induction with CoCl₂ or deferoxamine. Our results indicate that, although the expression of oxidoreductases shows some correlation with both the fluorescence signal from the hypoxia sensor and the cytotoxicity of evofosfamide, no correlation is observed between the hypoxia sensor readout and the prodrug’s activity. This implies that a single hypoxia marker may be insufficient for robust patient stratification and supports the need for a multiparametric framework that captures diverse aspects of the hypoxic tumor phenotype and activation biology of the prodrug.