Study on Sucrosomial® Iron Endocytosis-Mediated Uptake and Enterocytes Release
Authors: Brilli Elisa , Michela Asperti , Gryzik Magdalena , Lucchesi Alessandro , Martinelli Giovanni , Poli Maura , Tarantino Germano 
Published: October 2019

Abstract
Introduction: iron homeostasis is maintained by regulating the iron levels in plasma which is maintained by four coordinated processes: duodenal iron absorption, macrophage iron recycling, hepatic iron storage and erythropoiesis. Iron in the Fe2+ form is transported across the apical duodenal membrane by DMT1 and subsequently transferred to the blood via the iron exporter, Ferroportin the only know cell membrane iron exporter. Due to the presence of two check points at cellular levels, iron absorption and release are mainly regulated, because of this iron containing oral formulations are poorly absorbed and bioavailable. To overcome cellular barriers and increasing the bioavailability of supplemented iron forms, there is a need for new carriers that work protecting the iron as well as enhancing its intestinal absorption and release into the blood stream. Moreover thus reducing dosage and side effects. Sucrosomial® Iron (SI) represents an innovative oral iron-containing carrier in which ferric pyrophosphate is protected by a phospholipid bilayer membrane plus a sucrester matrix. To date, in vitro studies have shown that SI is mostly absorbed as vesicle-like structure, bypassing the conventional iron absorption pathway. Due to its behaviour at the gastrointestinal tract, SI is well tolerated and highly bioavailable compared to conventional iron salts. To deeply understand involvement of endocytosis in SI absorption and release, in vitro experiments using endocytosis and ferroportin inhibitors were carried out
Aim: to study Sucrosomial® Iron uptake and release in different in vitro systems.
Materials and Methods: CACO-2 and THP1 cells were used to investigate the role of FPN in Sucorsomial Iron release from cells. For release study, CACO-2 cells were exposed for 18h to quercetin (150mmol/L) in order to downregulate FPN expression. CACO-2 quercetin pre-treated cells were co-cultured with TPH1 cells, and SI or FAC were added. However, prior to measure cell Ferritin content, the incubation medium was discarded and cells were washed to remove quercetin. Iron uptake-release analysis was performed using co-culture transwell system between CACO-2 cells and TPH1. To investigate the cellular fate of cellular iron in quercetin treated CACO-2 and TPH1 cells we measured cell ferritin content. To inhibit endocytosis absorption pathway, CACO-2, THP1 and HepG2 cells were pre-treated with PitStop2 and Dyngo 4a inhibitors and then treated with SI, or Ferrous Sulfate (FS) or ferric ammonium citrate (FAC). Cellular Ferritin content was measured.
Results: in order to understand the effect of quercetin on iron storage, we used CACO2 and TPH1 cells pre-treated with quercetin and then treated with SI, FAC or nothing (control). Quercetin-SI treated CACO-2 cells showed no differences in Ferritin expression compared to control cells (3,94 ngFTL/mg proteins Vs 4,56 ngFTL/mg proteins) while in quercetin-FAC treated cells ferritin expression was decreased compare to control cells (16,3 ngFTL/mg proteins Vs 27,55 ngFTL/mg proteins). In a similar manner, quercetin-SI treated TPH1 cells didn't show increase in Ferritin expression compared to control cells (20 ngFTL/mg proteins Vs 15,15 ngFTL/mg proteins), only in quercetin-FAC treated cells we observed a Ferritin expression increase compared to control untreated cells (16 ngFTL/mg proteins Vs 24 ngFTL/mg proteins). Results from experiments using endocytosis inhibitors showed that SI absorption in CACO-2 cells is inhibited using Dyngo4a (from 4ngFTL/mg proteins to 0,36 ngFTL/ mg proteisn) while PitStop3 seems to reduce SI absorption in THP1 (from 396 ngFL/mg protein to 199,91 ngFTL/mg proteins) and HepG2 cells (from 26,86 ngFL/mg proteins to 3,93 ngFTL/mg proteins), since ferritin expression significantly decrease only in SI treated cells.
Conclusions: endocytosis pathway seems to be involved in SI cellular uptake but this process is regulated in different manner probably due to different cell types. Release experiments showed that cells treated with quercetin could reduce for a negative feedback DMT1 expression as well, affecting iron uptake from cells treated with FAC but not with SI and consequently, if SI is able to bypass commonly iron uptake mechanism, FPN inhibition did not show iron release perturbation from cells treated with SI.

Disclosures
Brilli: Pharmanutra s.p.a.: Consultancy. Martinelli:Janssen: Consultancy; Pfizer: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Roche: Consultancy; Abbvie: Consultancy; Novartis: Speakers Bureau; Amgen: Consultancy; Ariad/Incyte: Consultancy; Jazz Pharmaceuticals: Consultancy. Tarantino:Pharmanutra s.p.a.: Employment.