in-vitro-permeation-fitc-loaded-ferritins-across-rat-blood-brain

<em>In Vitro</em> Permeation of FITC-loaded Ferritins Across a Rat Blood-brain Barrier: a Model to Study the Delivery of Nanoformulated Molecules

Brain microvascular endothelial cells, supported by pericytes and astrocytes endfeet, are responsible for the low permeation of large hydrosoluble drugs ...

Università di Milano

Prior studies have suggested urinary oxygen tension (P(u)O2) as a putative index of renal blood flow (RBF) and tissue oxygenation. In the present study, we collected intraoperative data from eight anaemic, bladder-catheterized patients who received erythrocyte transfusions during various surgical procedures under general anaesthesia. Urinary and arterial blood gas analysis and co-oximetry were performed before and after transfusion, during an interval in which RBF was assumed to be constant. Pre- and post-transfusion haematocrit levels were 23 +/- 4.8 and 27 +/- 6.4%, respectively, and arterial oxygen content (C(a)O2) was 10.5 +/- 2.5 and 12.4 +/- 3.1 mL/dL, respectively (P = 0.012). Pre- and post-transfusion P(u)O2 was 90 +/- 14 and 108 +/- 20 mmHg, respectively (P = 0.036). 4. These results, although limited, suggest that under conditions of stable haemodynamics, systemic oxygenation and renal function, intraoperative blood transfusion may increase P(u)O2 in anaemic anaesthetized patients. If confirmed by subsequent clinical and laboratory studies, P(u)O2 measurement could become a simple, non-invasive way to monitor renal medullary oxygenation and tissue oxygen availability and help determine whether red blood cells should be transfused.

Post-transfusional variation in urinary oxygen tension in surgical patients.

Achieving clear margins with adequate resection volumes is one of the principal goals of breast-conserving surgery. The aim of our study was to compare preoperative localization using 2 different clips, radiopaque or sonographically visible, to reach this goal.

Localization of nonpalpable breast lesions with sonographically visible clip: optimizing tailored resection and clear margins.

The receptor for the advanced glycation end products (RAGE) is a type I transmembrane glycoprotein belonging to the immunoglobulin superfamily and binds a variety of unrelated ligands sharing a negative charge. Most ligands bind to the extracellular V or VC1 domains of the receptor. In this work, V and VC1 of human RAGE were produced in the methylotrophic yeast Pichia pastoris and directed to the secretory pathway. Fusions to a removable C-terminal His-tag evidenced proteolytic processing of the tag by extracellular proteases and also intracellular degradation of the N-terminal portion of V-His. Expression of untagged forms was attempted. While the V domain was retained intracellularly, VC1 was secreted into the medium and was functionally active in binding AGEs. The glycosylation state of VC1 was analyzed by mass spectrometry and peptide-N-glycosidase F digestion. Like RAGE isolated from mammalian sources, the degree of occupancy of the N-glycosylation sites was full at Asn25 and partial at Asn81 which was also subjected to non-enzymatic deamidation. A simple procedure for the purification to homogeneity of VC1 from the medium was developed. The folded state of the purified protein was assessed by thermal shift assays. Recombinant VC1 from P. pastoris showed a remarkably high thermal stability as compared to the protein expressed in bacteria. Our in vivo approach indicates that the V and C1 domains constitute a single folding unit. The stability and solubility of the yeast-secreted VC1 may be beneficial for future in vitro studies aimed to identify new ligands or inhibitors of RAGE.

An improved expression system for the VC1 ligand binding domain of the receptor for advanced glycation end products in Pichia pastoris.

Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein with pro-life and pro-death activities, which plays critical roles in mitochondrial energy metabolism and caspase-independent apoptosis. Defects in AIF structure or expression can cause mitochondrial abnormalities leading to mitochondrial defects and neurodegeneration. The mechanism of AIF-induced apoptosis was extensively investigated, whereas the mitochondrial function of AIF is poorly understood. A unique feature of AIF is the ability to form a tight, air-stable charge-transfer (CT) complex upon reaction with NADH and to undergo a conformational switch leading to dimerization, proposed to be important for its vital and lethal functions. Although some aspects of interaction of AIF with NAD(+)/H have been analyzed, its precise mechanism is not fully understood. We investigated how the oxidized and photoreduced wild-type and G307A and -E variants of murine AIF associate with NAD(+)/H and nicotinamide mononucleotide (NMN(+)/H) to determine the role of the adenylate moiety in the binding process. Our results indicate that (i) the adenylate moiety of NAD(+)/H is crucial for the association with AIF and for the subsequent structural reorganization of the complex, but not for protein dimerization, (ii) FAD reduction rather than binding of NAD(+)/H to AIF initiates conformational rearrangement, and (iii) alteration of the adenylate-binding site by the G307E (equivalent to a pathological G308E mutation in human AIF) or G307A replacements decrease the affinity and association rate of NAD(+)/H, which, in turn, perturbs CT complex formation and protein dimerization but has no influence on the conformational switch in the regulatory peptide.

Key Role of the Adenylate Moiety and Integrity of the Adenylate-Binding Site for the NAD(+)/H Binding to Mitochondrial Apoptosis-Inducing Factor.

Nowadays cancer represents a prominent challenge in clinics. Main achievements in cancer management would be the development of highly accurate and specific diagnostic tools for early detection of cancer onset, and the generation of smart drug delivery systems for targeted chemotherapy release in cancer cells. In this context, protein-based nanocages hold a tremendous potential as devices for theranostics purposes. In particular, ferritin has emerged as an excellent and promising protein-based nanocage thanks to its unique architecture, surface properties and high biocompatibility. By exploiting natural recognition of the Transferrin Receptor 1, which is overexpressed on tumor cells, ferritin nanocages may ensure a proper drug delivery and release. Moreover, researchers have applied surface functionalities on ferritin cages for further providing active tumor targeting. Encapsulation strategies of non metal-containing drugs within ferritin cages have been explored and successfully performed with encouraging results. Various preclinical studies have demonstrated that nanoformulation within ferritin nanocages significantly improved targeted therapy and accurate imaging of cancer cells. Aims of this review are to describe structure and functions of ferritin nanocages, and to provide an overview about the nanotechnological approaches implemented for applying them to cancer diagnosis and treatment.

Ferritin nanocages: A biological platform for drug delivery, imaging and theranostics in cancer.

In this study, insulin-containing nanoparticles were loaded into pellet cores and orally administered to diabetic rats. Polyethylene imine-based nanoparticles, either placebo or loaded with insulin, were incorporated by extrusion and spheronization technology into cores that were subsequently coated with three overlapping layers and a gastroresistant film. The starting and coated systems were evaluated in vitro for their physico-technololgical characteristics, as well as disintegration and release performance. Nanoparticles-loaded cores showed homogeneous particle size distribution and shape. When a superdisintegrant and a soluble diluent were included in the composition enhanced disintegration and release performance were observed. The selected formulations, coated either with enteric or three-layer films, showed gastroresistant and release delayed behavior in vitro, respectively. The most promising formulations were finally tested for their hypoglycemic effect in diabetic rats. Only the nanoformulations loaded into the three-layer pellets were able to induce a significant hypoglycemic activity in diabetic rats. Our results suggest that this efficient activity could be attributed to a retarded release of insulin into the distal intestine, characterized by relatively low proteolytic activity and optimal absorption.

Luca Sorrentino

Dipartimento di Scienze Biomediche e Cliniche Luigi Sacco

Università di Milano

In Vitro Permeation of FITC-loaded Ferritins Across a Rat Blood-brain Barrier: a Model to Study the Delivery of Nanoformulated Molecules