Analytical strategies to study the formation and drug delivery capabilities of ferritin-encapsulated cisplatin in sensitive and resistant cell models.

One of the limitations in the use of cisplatin is its low penetration into cells. In addition, some cells develop the so called resistance, a multifactorial event that decreases significantly the intracellular cisplatin concentration. To circumvent these limitations, recent studies are focused on the use of nanocarriers that permit, among others, to achieve higher drug uptake. In this work, ferritin is evaluated as a nanostructured cisplatin-delivery system in cell models of ovarian cancer. One of the key aspects is the characterization of the encapsulated product, and for this aim, a battery of analytical techniques, including size exclusion chromatography (SEC) coupled to UV detection and to inductively coupled plasma mass spectrometry (ICP-MS) together with transmission electron microscopy (TEM), is conducted. Higher level of incorporation occurs when using initial concentrations of the Fe-containing form of the protein at 10 mg/mL and 1 mg/mL cisplatin solution. The incorporation of the free and encapsulated cisplatin is addressed in A2780 and A2780CIS, sensitive and cisplatin-resistant cell lines, respectively, showing a significantly higher uptake of the encapsulated form. These values ranged from 5- to 9-fold in the sensitive line and 2-4 in the resistant model, being always more pronounced at the lower doses. Functionality of the drug after encapsulation is addressed by monitoring the presence of Pt in DNA and normalizing DNA concentration through simultaneous P and Pt measurements by ICP-MS. Time elapsed between exposure and Pt detection in DNA proved to be critical in the encapsulated model, showing the slower drug release mechanism from the ferritin nanocage that could be advantageously used for a controlled therapy. Graphical abstract.

Gadolinium released by the linear gadolinium-based contrast-agent Gd-DTPA decreases the activity of human epithelial Na+ channels (ENaCs).

BACKGROUND: Gadolinium-based-contrast-agents (GBCAs) are used for magnetic-resonance-imaging and associated with renal and cardiovascular adverse reactions caused by released Gd3+ ions. Gd3+ is also a modulator of mechano-gated ion channels, including the epithelial Na+ channel (ENaC) that is expressed in kidney epithelium and the vasculature. ENaC is important for salt-/water homeostasis and blood pressure regulation and a likely target of released Gd3+ from GBCAs causing the above-mentioned adverse reactions. Therefore this study examined the effect of Gd3+ and GBCAs on ENaC's activity. METHODS: Human αßγENaC was expressed in Xenopus laevis oocytes and exposed to Gd3+, linear (Gd-DTPA, Magnevist) or cyclic (Dotarem) GBCAs. Transmembrane ion-currents (IM) were recorded by the two-electrode-voltage-clamp technique and Gd3+-release by Gd-DTPA was confirmed by inductively coupled plasma-mass spectrometry. RESULTS: Gd3+ exerts biphasic effects on ENaC's activity: ≤0.3mmol/l decreased IM which was preventable by DEPC (modifies histidines). Strikingly Gd3+≥0.4mmol/l increased IM and this effect was prevented by cysteine-modifying MTSEA. Linear Gd-DTPA and Magnevist mimicked the effect of ≤0.3mmol/l Gd3+, whereas the chelator DTPA showed no effect. Gd3+ and Gd-DTPA increased the IC50 for amiloride, but did not affect ENaC's self-inhibition. Interestingly, cyclic Gd-DOTA (Dotarem) increased IM to a similar extent as its chelator DOTA, suggesting that the chelator rather than released Gd3+ is responsible for this effect. CONCLUSION: These results confirm Gd3+-release from linear Gd-DTPA and indicate that the released Gd3+ amount is sufficient to interfere with ENaC's activity to provide putative explanations for GBCA-related adverse effects.