Doxorubicin loaded octacalcium phosphate particles as controlled release drug delivery systems: Physico-chemical characterization, in vitro drug release and evaluation of cell death pathway.

Mastering new and efficient ways to obtain successful drug delivery systems (DDS) with controlled release became a paramount quest in the scientific community. Increase of malignant bone tumors and the necessity to optimize an approach of localized drug delivery require research to be even more intensified. Octacalcium phosphate (OCP), with a number of advantages over current counterparts is extensively used in bone engineering. The aim of the present research was to synthesize bioactive and biocompatible doxorubicin (DOX) containing OCP particles. DOX-OCP was successfully obtained in situ in an exhaustive range of added drug (1-20 wt%, theoretical loading). Based on XRD, above 10 wt% of DOX, OCP formation was inhibited and the obtained product was low crystalline α-TCP. In-vitro drug release was performed in pH 7.4 and 6.0. In both pH environments DOX had a continuous release over six weeks. However, the initial drug burst for pH 7.4, in the first 24 h, ranged from 15.9 ± 1.3 % to 33.5 ± 12 % and for pH 6.0 23.7 ± 1.5 % to 36.2 ± 12 %.The DOX-OCP exhibited an inhibitory effect on viability of osteosarcoma cell lines MG63, U2OS and HOS. In contrast, MC3T3-E1 cells (IC50 > 0.062 µM) displayed increased viability and proliferation from 3rd to 7th day. Testing of the DDS on ferroptotic markers (CHAC1, ACSL4 and PTGS2) showed that OCP-DOX does not induce ferroptotic cell death. Moreover, the evaluation of protein levels of cleaved PARP, by western blotting analysis, corroborated that apoptosis is the main pathway of programmed cell death in osteosarcoma cells induced by DOX-OCP.

FSP1 is a predictive biomarker of osteosarcoma cells' susceptibility to ferroptotic cell death and a potential therapeutic target.

Human osteosarcoma (OS) is a relatively rare malignancy preferentially affecting long body bones which prognosis is often poor also due to the lack of effective therapies. Clinical management of this cancer basically relies on surgical removal of primary tumor coupled with radio/chemotherapy. Unfortunately, most osteosarcoma cells are resistant to conventional therapy, with the undergoing epithelial-mesenchymal transition (EMT) giving rise to gene expression reprogramming, thus increasing cancer cell invasiveness and metastatic potential. Alternative clinical approaches are thus urgently needed. In this context, the recently described ferroptotic cell death represents an attractive new strategy to efficiently kill cancer cells, since most chemoresistant and mesenchymal-shaped tumors display high susceptibility to pro-ferroptotic compounds. However, cancer cells have also evolved anti-ferroptotic strategies, which somehow sustain their survival upon ferroptosis induction. Indeed, here we show that osteosarcoma cell lines display heterogeneous sensitivity to ferroptosis execution, correlating with the mesenchymal phenotype, which is consistently affected by the expression of the well-known anti-ferroptotic factor ferroptosis suppressor protein 1 (FSP1). Interestingly, inhibiting the activity or expression of FSP1 restores cancer cell sensitivity to ferroptosis. Moreover, we also found that: i) AKRs might also contribute to resistance; ii) NRF2 enhances FSP1 expression upon ferroptosis induction; while iii) p53 contributes to the regulation of FSP1 basal expression in OS cells.In conclusion, FSP1 expression can potentially be used as a valuable predictive marker of OS sensitivity to ferroptosis and as a new potential therapeutic target.

The Gut-Ex-Vivo System (GEVS) Is a Dynamic and Versatile Tool for the Study of DNBS-Induced IBD in BALB/C and C57BL/6 Mice, Highlighting the Protective Role of Probiotics.

BACKGROUND: IBD is a spectrum of pathologies characterized by dysregulated immune activation leading to uncontrolled response against the intestine, thus resulting in chronic gut inflammation and tissue damage. Due to its complexity, the molecular mechanisms responsible for disease onset and progression are still elusive, thus requiring intense research effort. In this context, the development of models replicating the etiopathology of IBD and allowing the testing of new potential therapies is critical. METHODS: Colon from C57BL/6 or BALB/c mice was cultivated in a Gut-Ex-Vivo System (GEVS), exposed for 5 h to DNBS 1.5 or 2.5 mg/mL, in presence or absence of two probiotic formulations (P1 = Bifidobacterium breve BR03 (DSM16604) and B632 (DSM24706); P2 = Lacticaseibacillus rhamnosus LR04 (DSM16605), Lactiplantibacillus plantarum LP14 (DSM33401) and Lacticaseibacillus paracasei LPC09), and the main hallmarks of IBD were evaluated. RESULTS: Gene expression analysis revealed the following DNBS-induced effects: (i) compromised tight junction organization, responsible for tissue permeability dysregulation; (ii) induction of ER stress, and (iii) tissue inflammation in colon of C57BL/6 mice. Moreover, the concomitant DNBS-induced apoptosis and ferroptosis pathways were evident in colon from both BALB/c and C57BL/6 mice. Finally, the co-administration of probiotics completely prevented the detrimental effects of DNBS. CONCLUSIONS: Overall, we have provided results demonstrating that GEVS is a consistent, reliable, and cost-effective system for modeling DNBS-induced IBD, useful for studying the onset and progression of human disease at the molecular level, while also reducing animal suffering. Moreover, we have confirmed the beneficial effect of probiotics administration in promoting the remission of IBD.

A Gut-Ex-Vivo System to Study Gut Inflammation Associated to Inflammatory Bowel Disease (IBD).

Inflammatory bowel disease (IBD) is a complex, chronic, and dysregulated inflammatory condition which etiology is still largely unknown. Its prognosis and disease progression are highly variable and unpredictable. IBD comprises several heterogeneous inflammatory conditions ranging from Ulcerative Colitis (UC) to Crohn's Disease (CD). Importantly, a definite, well-established, and effective clinical treatment for these pathologies is still lacking. The urgent need for treatment is further supported by the notion that patients affected by UC or CD are also at risk of developing cancer. Therefore, a deeper understanding of the molecular mechanisms at the basis of IBD development and progression is strictly required to design new and efficient therapeutic regimens. Although the development of animal models has undoubtedly facilitated the study of IBD, such in vivo approaches are often expensive and time-consuming. Here we propose an organ ex vivo culture (Gut-Ex-Vivo system, GEVS) based on colon from Balb/c mice cultivated in a dynamic condition, able to model the biochemical and morphological features of the mouse models exposed to DNBS (5-12 days), in 5 h. Indeed, upon DNBS exposure, we observed a dose-dependent: (i) up-regulation of the stress-related protein transglutaminase 2 (TG2); (ii) increased intestinal permeability associated with deregulated tight junction protein expression; (iii) increased expression of pro-inflammatory cytokines, such as TNFα, IFNγ, IL1ß, IL6, IL17A, and IL15; (iv) down-regulation of the anti-inflammatory IL10; and (v) induction of Endoplasmic Reticulum stress (ER stress), all markers of IBD. Altogether, these data indicate that the proposed model can be efficiently used to study the pathogenesis of IBD, in a time- and cost-effective manner.

Probiotics Supplements Reduce ER Stress and Gut Inflammation Associated with Gliadin Intake in a Mouse Model of Gluten Sensitivity.

Exposure to gluten, a protein present in wheat rye and barley, is the major inducer for human Celiac Disease (CD), a chronic autoimmune enteropathy. CD occurs in about 1% worldwide population, in genetically predisposed individuals bearing human leukocyte antigen (HLA) DQ2/DQ8. Gut epithelial cell stress and the innate immune activation are responsible for the breaking oral tolerance to gliadin, a gluten component. To date, the only treatment available for CD is a long-term gluten-free diet. Several studies have shown that an altered composition of the intestinal microbiota (dysbiosis) could play a key role in the pathogenesis of CD through the modulation of intestinal permeability and the regulation of the immune system. Here, we show that gliadin induces a chronic endoplasmic reticulum (ER) stress condition in the small intestine of a gluten-sensitive mouse model and that the coadministration of probiotics efficiently attenuates both the unfolded protein response (UPR) and gut inflammation. Moreover, the composition of probiotics formulations might differ in their activity at molecular level, especially toward the three axes of the UPR. Therefore, probiotics administration might potentially represent a new valuable strategy to treat gluten-sensitive patients, such as those affected by CD.

Gut-Ex-Vivo system as a model to study gluten response in celiac disease.

Celiac disease (CD) is a complex immune-mediated chronic disease characterized by a consistent inflammation of the gastrointestinal tract induced by gluten intake in genetically predisposed individuals. Although initiated by the interaction between digestion-derived gliadin, a gluten component, peptides, and the intestinal epithelium, the disorder is highly complex and involving other components of the intestine, such as the immune system. Therefore, conventional model systems, mainly based on two- or three-dimension cell cultures and co-cultures, cannot fully recapitulate such a complex disease. The development of mouse models has facilitated the study of different interacting cell types involved in the disorder, together with the impact of environmental factors. However, such in vivo models are often expensive and time consuming. Here we propose an organ ex vivo culture (gut-ex-vivo system) based on small intestines from gluten-sensitive mice cultivated in a dynamic condition, able to fully recapitulate the biochemical and morphological features of the mouse model exposed to gliadin (4 weeks), in 16 h. Indeed, upon gliadin exposure, we observed: i) a down-regulation of cystic fibrosis transmembrane regulator (CFTR) and an up-regulation of transglutaminase 2 (TG2) at both mRNA and protein levels; ii) increased intestinal permeability associated with deregulated tight junction protein expression; iii) induction and production of pro-inflammatory cytokines such as interleukin (IL)-15, IL-17 and interferon gamma (IFNγ); and iv) consistent alteration of intestinal epithelium/villi morphology. Altogether, these data indicate that the proposed model can be efficiently used to study the pathogenesis of CD, test new or repurposed molecules to accelerate the search for new treatments, and to study the impact of the microbiome and derived metabolites, in a time- and cost- effective manner.

Gellan gum hydrogels cross-linked with carbodiimide stimulates vacuolation of human tooth-derived stem cells in vitro.

The natural polysaccharides are promising compounds for applications in regenerative medicine. Gellan gum (GG) is the bacteria-derived polysaccharide widely used in food industry. Simple modifications of its chemical properties make GG superior for the development of biocompatible hydrogels. Beside reversible cationic integration of GG chains, more efficient binding is accomplished with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC). However, the side-products of polymer cross-linking might affect viability and differentiation of stem cells introduced into the hydrogels. We found that O-acylisourea (EDU) stimulates autophagy-based vacuolation in both periodontal ligament and dental pulp stem cells. 24-h treatment of cells with GG extracts cross-linked with 15 mM EDC developed large cytoplasmic vacuoles. Freshly prepared EDU (2-6 mM) but not 15 mM EDC solutions initiated vacuole development with concomitant reduction of cell viability/metabolism. Most of the vacuoles stained with acridine orange displayed highly acidic environment further confirmed by flow cytometric analysis. Western blot of the LC3 autophagy marker followed by a transmission electron microscopy indicated the process is autophagy-dependent. We propose that the high reactivity of EDU with intracellular components initiates autophagy, although the targets of EDU remain unknown. Nevertheless, a burst release of EDU from GG hydrogels might modulate negatively cellular processes and final effectiveness of tissue regeneration.