366. Development of Engineered Magnetic Nanoparticles Coupled With Lentiviral Vectors for Targeted Cancer Therapy and Hyperthermia

BackgroundIn recent years, considerable efforts have been spent to develop magnetic nanoparticles (MNPs) and to improve their applicability in several areas including hyperthermia and target cancer gene therapies.The aim of the present study was to synthesize and characterize Fe3O4, magnetite core-silica shell and magnetite core-silica shell doped with calcium ions nanoparticles (NPs) in combination with lentiviral vectors to deliver therapeutic genes in vivo.Materials and MethodsMagnetite NPs were prepared by co-precipitation method, the silica shell was obtained by wet chemistry on the magnetic core stabilized with citric acid. Calcium ions were added to the silica shell modulating the NPs surface reactivity.The NPs were characterized with X-Ray diffraction, transmission electron microscopy, Vibrating Sample Magnetometer and zeta potential.Cytocompatibility tests were performed using both direct and not-direct contact models with murine endothelial cells (MS1) both in static and dynamic conditions using MNP coupled with/out LV.MNP and MNP-LV were tail vein injected intravenously in C57/Bl6 mice, biodistribution and expression studies were performed by histology and immunofluorescence using GFP as a marker gene.ResultsSpherical magnetite nanoparticles of about 15 nm in diameter were obtained with good dispersion in water. Addition of silica and calcium allowed obtaining a thin and amorphous silica or Ca-enriched silica shell, maintaining good dispersion in water. All the MNPs displayed a superparamagnetic behaviour.The MNP+/-LV used demonstrated to be cytocompatible in both static traditional and dynamic cytocompatibility models. Moreover when MNP-LV injected in mice we detected GFP expression mainly in the liver and spleen with biodistribution differences based on the MNPs-LV combination used.ConclusionThese results suggest these NPs as promising for in vivo applications. Biodistribution studies in vivo of Fe3O4 NPs in mice models were performed and accumulation of NPs into vital organs was minimal with no toxicity in mice up to 1 month later and sustained GFP expression detected with no inflammatory responses. The present studies can significantly improve the cancer therapy effectiveness by means of a selective and localized delivery of transgenes together with the opportunity to conjugate hyperthermic and genetic approaches using therapeutic transgenes

Innovative superparamagnetic iron-oxide nanoparticles coated with silica and conjugated with linoleic acid: Effect on tumor cell growth and viability

One of the goals for the development of more effective cancer therapies with reduced toxic side effects is the optimization of innovative treatments to selectively kill tumor cells. The use of nanovectors loaded with targeted therapeutic payloads is one of the most investigated strategies. In this paper superparamagnetic iron oxide nanoparticles (SPIONs) coated by a silica shell or uncoated, were functionalized with single-layer and bi-layer conjugated linoleic acid (CLA). Silica was used to protect the magnetic core from oxidation, improve the stability of SPIONs and tailor their surface reactivity. CLA was used as novel grafting biomolecule for its anti-tumor activity and to improve particle dispersibility. Mouse breast cancer 4T1 cells were treated with these different SPIONs. SPIONs functionalized with the highest quantity of CLA and coated with silica shell were the most dispersed. Cell viability was reduced by SPIONs functionalized with CLA in comparison with cells which were untreated or treated with SPIONs without CLA. As regards the types of SPIONs functionalized with CLA, the lowest viability was observed in cells treated with uncoated SPIONs with the highest quantity of CLA. In conclusion, the silica shell free SPIONs functionalized with the highest amount of CLA can be suggested as therapeutic carriers because they have the best dispersion and ability to decrease 4T1 cell viability

Therapeutic potential of fetal liver cells transplantation in hemophilia A mice

: Hemophilia A (HA) cell therapy approaches in pediatric individuals require suitable factor (F)VIII-producing cells for stable engraftment. Liver sinusoidal endothelial cells (LSEC) and hematopoietic stem cells (HSC) have been demonstrated to be suitable for the treatment of adult HA-mice. However, after transplantation in busulfan (BU)-conditioned newborn mice, adult LSEC/HSC cannot efficiently engraft, while murine fetal liver (FL) hemato/vascular cells from embryonic day 11-13 of gestation (E11-E13), strongly engraft the hematopoietic and endothelial compartments while also secreting FVIII. Our aim was to investigate the engraftment of FL cells in newborn HA mice for obtaining a suitable "proof of concept" for the development of a new HA treatment in neonates. Hence, we transplanted FLE11 or E13 cells and adult bone marrow (BM) cells into newborn HA mice with or without BU preconditioning. The engraftment levels and FVIII activity was assessed starting from 6 weeks after transplantation. FLE11-E13+BU-transplanted newborns reached up to 95% engraftment with stable FVIII activity levels observed for 16 months. FLE13 cells showed engraftment ability even in absence of BU preconditioning, while FLE11 cells did not. BM+BU transplanted newborn HA mice showed high levels of engraftment; nevertheless, in contrast to FL cells, BM cells cannot engraft HA newborns in non-conditioning regimen. Finally, none of the transplanted mice developed anti-FVIII antibodies. Overall, this study sheds some light on the therapeutic potential of healthy FL cells in the cure of HA neonatal/pediatric patients

Innovative superparamagnetic iron-oxide nanoparticles coated with silica and conjugated with linoleic acid: Effect on tumor cell growth and viability

One of the goals for the development of more effective cancer therapies with reduced toxic side effects is the optimization of innovative treatments to selectively kill tumor cells. The use of nanovectors loaded with targeted therapeutic payloads is one of the most investigated strategies. In this paper superparamagnetic iron oxide nanoparticles (SPIONs) coated by a silica shell or uncoated, were functionalized with single-layer and bi-layer conjugated linoleic acid (CLA). Silica was used to protect the magnetic core from oxidation, improve the stability of SPIONs and tailor their surface reactivity. CLA was used as novel grafting biomolecule for its anti-tumor activity and to improve particle dispersibility. Mouse breast cancer 4T1 cells were treated with these different SPIONs. SPIONs functionalized with the highest quantity of CLA and coated with silica shell were the most dispersed. Cell viability was reduced by SPIONs functionalized with CLA in comparison with cells which were untreated or treated with SPIONs without CLA. As regards the types of SPIONs functionalized with CLA, the lowest viability was observed in cells treated with uncoated SPIONs with the highest quantity of CLA. In conclusion, the silica shell free SPIONs functionalized with the highest amount of CLA can be suggested as therapeutic carriers because they have the best dispersion and ability to decrease 4T1 cell viability

Lentiviral vector interactions with the host cell

Lentiviral vectors (LVs)-mediated gene transfer is an efficient method for ex vivo and in vivo gene therapy. Actually, LVs have been used in several clinical trials and therapeutic correction was reached in affected patients. However, in order to be effective gene therapy needs to be efficient without detrimental effects for target cells. Successful cell transduction by LVs can be hampered by several factors such as the activation of innate immune sensors during cell transduction and different restriction factors (RFs) inhibiting viral replication inside the cells. Therefore, a better knowledge of host-vector interactions is important for the development of more efficient gene therapy strategies improving the LVs platform by limiting harmful responses

Il Convegno nazionale di CNAI 2014 con NURSES FOR EXPO ispirato dal tema di EXPO Milano 2015: Nutrire il pianeta, energia per la vita

Il Convegno Nazionale CNAI (Consociazione Nazionale delle associazioni infermiere/i), svoltosi a Bergamo presso l'Auditorium della Casa del giovane nei giorni 9 e 10 ottobre 2014, aveva come tema il ruolo infermieristico a favore dell'alimentazione e della salute. Il convegno era collocato in una serie di iniziative che vedranno coinvolti gli infermieri italiani nel progetto NURSES FOR EXPO lanciato in occasione dell'EXPO che vedrí  impegnata Milano, e non solo, in prima linea nell'anno 2015 sul tema Nutrire il pianeta, energia per la vita. Il desiderio di far conoscere il ruolo dell'infermiere in questo ambito ha portato alla creazione di un logo per questo progetto, con l'intento di diffondere questa iniziativa, avvalendosi, come consigliato dalla presidente CNAI Cecilia Sironi, dei social network e contando soprattutto sui futuri infermieri, presenti in un buon numero al Convegno, provenienti da diverse universití  lombarde. Durante le due giornate sono intervenuti circa 20 infermieri, giunti da diverse Regioni e da diverse realtí  lavorative, portando esperienze legate sia all'attivití  quotidiana nei reparti, sia a ricerche scientifiche svolte negli ultimi anni

Immune tolerance promotion by LSEC-specific lentiviral vector-mediated expression of the transgene regulated by the stabilin-2 promoter

Liver sinusoidal endothelial cells (LSECs) are specialized endocytic cells that clear the body from blood-borne pathogens and waste macromolecules through scavenger receptors (SRs). Among the various SRs expressed by LSECs is stabilin-2 (STAB2), a class H SR that binds to several ligands, among which endogenous coagulation products. Given the well-established tolerogenic function of LSECs, we asked whether the STAB2 promoter (STAB2p) would enable us to achieve LSEC-specific lentiviral vector (LV)-mediated transgene expression, and whether the expression of this transgene would be maintained over the long term due to tolerance induction. Here, we show that STAB2p ensures LSEC-specific green fluorescent protein (GFP) expression by LV in the absence of a specific cytotoxic CD8+ T cell immune response, even in the presence of GFP-specific CD8+ T cells, confirming the robust tolerogenic function of LSECs. Finally, we show that our delivery system can partially and permanently restore FVIII activity in a mouse model of severe hemophilia A without the formation of anti-FVIII antibodies. Overall, our findings establish the suitability of STAB2p for long-term LSEC-restricted expression of therapeutic proteins, such as FVIII, or to achieve antigen-specific immune tolerance in auto-immune diseases