Giovanni Maria Severini

NIR-labeled nanoparticles engineered for brain targeting: in vivo optical imaging application and fluorescent microscopy evidences

The presence of the blood–brain barrier (BBB) makes extremely difficult to develop efficacious strategies for targeting contrast agents and delivering drugs inside the Central Nervous System (CNS). To overcome this drawback, several kinds of CNS-targeted nanoparticles (NPs) have been developed. In particular, we proposed poly-lactide-co-glycolide (PLGA) NPs engineered with a simil-opioid glycopeptide (g7), which have already proved to be a promising tool for achieving a successful brain targeting after i.v. administration in rats. In order to obtain CNS-targeted NPs to use for in vivo imaging, we synthesized and administrated in mice PLGA NPs with double coverage: near-infrared (NIR) probe (DY-675) and g7. The optical imaging clearly showed a brain localization of these novel NPs. Thus, a novel kind of NIR-labeled NPs were obtained, providing a new, in vivo detectable nanotechnology tool. Besides, the confocal and fluorescence microscopy evidences allowed to further confirm the ability of g7 to promote not only the rat, but also the mouse BBB crossing.

Reversal of Diabetes in Mice by Implantation of Human Fibroblasts Genetically Engineered to Release Mature Human Insulin

Autoimmune destruction of pancreatic beta cells in type I, insulin-dependent diabetes mellitus (IDDM) results in the loss of endogenous insulin secretion, which is incompletely replaced by exogenous insulin administration. The functional restoration provided by allogeneic beta-cell transplantation is limited by adverse effects of immunosuppression. To pursue an insulin replacement therapy based on autologous, engineered human non-beta cells, we generated a retroviral vector encoding a genetically modified human proinsulin, cleavable to insulin in non-beta cells, and a human nonfunctional cell surface marker. Here we report that this vector efficiently transduced primary human cells, inducing the synthesis of a modified proinsulin that was processed and released as mature insulin. This retrovirally derived insulin displayed in vitro biological activity, specifically binding to and phosphorylation of the insulin receptor, comparable to human insulin. In vivo, the transplantation of insulin-producing fibroblasts reverted hyperglycemia in a murine model of diabetes, whereas proinsulin-producing cells were ineffective. These results support the possibility of developing insulin production machinery in human non-beta cells for gene therapy of IDDM.

Low frequency of detection by nested polymerase chain reaction of enterovirus ribonucleic acid in endomyocardial tissue of patients with idiopathic dilated cardiomyopathy

OBJECTIVESnThe purpose of this study was to determine the prevalence of enteroviral infection in the myocardium of patients with idiopathic dilated cardiomyopathy by using a highly sensitive and specific detection technique.nnnBACKGROUNDnRecent molecular studies have suggested that enteroviral persistence (in particular, coxsackieviruses type B) may underlie idiopathic myocarditis and dilated cardiomyopathy.nnnMETHODSnThe method used to detect enterovirus-specific ribonucleic acids (RNAs) is based on reverse transcription and nested polymerase chain reaction amplification with four pairs of primers from the conserved 5 noncoding region of the enteroviral genome. Several members of the Enterovirus genus are detectable by this assay (coxsackieviruses B1 to B6; polioviruses 1 to 3; echoviruses 9, 19 and 31), with a sensitivity threshold close to the detection of a single molecule of viral RNA in 1 mg of tissue sample. Endomyocardial tissue samples from 84 subjects were analyzed (77 samples obtained from left endomyocardial biopsies, 7 from explanted hearts). The subjects comprised 63 study patients (53 with dilated cardiomyopathy, 3 with idiopathic myocarditis, 1 with right ventricular dysplasia, 1 with restrictive cardiomyopathy, 1 with eosinophilic myocarditis, 1 with primary ventricular fibrillation and 3 with myocarditis of known etiology) and 21 control subjects with other diseases.nnnRESULTSnPositive signals were obtained only in samples from six study patients (four with dilated cardiomyopathy, one with right ventricular dysplasia and one with myocarditis). Samples from control subjects, uninfected rat myocardium and cultured cell lines yielded systematically negative results. Moreover, the nucleotide sequence analysis of the amplification products from patients with positive samples raised doubts about the true positivity of these samples.nnnCONCLUSIONSnThis study suggests that the persistence of enteroviral RNA in dilated cardiomyopathy is not a major cause of the disease and that a careful analysis of polymerase chain reaction amplification products is essential in any study in which this technique is pushed to high sensitivity thresholds.

Potential Use of Polymeric Nanoparticles for Drug Delivery Across the Blood-Brain Barrier

Nanomedicine is certainly one of the scientific and technological challenges of the coming years. In particular, biodegradable nanoparticles formulated from poly (D,L-lactide-co-glycolide) (PLGA) have been extensively investigated for sustained and targeted delivery of different agents, including recombinant proteins, plasmid DNA, and low molecular weight compounds. PLGA NPs present some very attractive properties such as biodegradability and biocompatibility, protection of drug from degradation, possibility of sustained release, and the possibility to modify surface properties to target nanoparticles to specific organs or cells. Moreover, PLGA NPs have received the FDA and European Medicine Agency approval in drug delivery systems for parenteral administration, thus reducing the time for human clinical applications. This review in particular deals on surface modification of PLGA NPs and their possibility of clinical applications, including treatment for brain pathologies such as brain tumors and Lysosomal Storage Disorders with neurological involvement. Since a great number of pharmacologically active molecules are not able to cross the Blood-Brain Barrier (BBB) and reach the Central Nervous System (CNS), new brain targeted polymeric PLGA NPs modified with glycopeptides (g7- NPs) have been recently produced. In this review several in vivo biodistribution studies and pharmacological proof-of evidence of brain delivery of model drugs are reported, demonstrating the ability of g7-NPs to create BBB interaction and trigger an efficacious BBB crossing. Moreover, another relevant development of NPs surface engineering was achieved by conjugating to the surface of g7-NPs, some specific and selective antibodies to drive NPs directly to a specific cell type once inside the CNS parenchyma.

Cell-based therapies for diabetic complications

In recent years, accumulating experimental evidence supports the notion that diabetic patients may greatly benefit from cell-based therapies, which include the use of adult stem and/or progenitor cells. In particular, mesenchymal stem cells and the circulating pool of endothelial progenitor cells have so far been the most studied populations of cells proposed for the treatment of vascular complications affecting diabetic patients. We review the evidence supporting their use in this setting, the therapeutic benefits that these cells have shown so far as well as the challenges that cell-based therapies in diabetic complications put out.

Molecular genetics of dilated cardiomyopathy

A major advance in the study of the pathogenesis of dilated cardiomyopathy (DC) has been the identification of a familial trait in a relevant proportion of cases (more than 25%), which indicates that, at least in these cases, a mutated gene is the cause of the disease. Familial dilated cardiomyopathy is a genetically heterogeneous disorder, most frequently with autosomal-dominant inheritance. Five different loci that cosegregate with the disease have been mapped so far; the identification of the disease genes is still in progress. The only disease gene known so far is the dystrophin gene, which causes X-linked DC. By analogy with dystrophin, it is believed that other cytoskeletal proteins could be involved in the pathogenesis of DC. Finally, in right ventricular cardiomyopathy, a peculiar form of cardiomyopathy that is frequently familial as well, several disease loci have been described. Also in this case, no disease gene has been yet identified. The advances in clinical and molecular genetics of DC have relevant clinical and therapeutic implications.

Various Cells Retrovirally Transduced with N-Acetylgalactosoamine-6-Sulfate Sulfatase Correct Morquio Skin Fibroblasts In Vitro

Gene therapy may provide a long-term approach to the treatment of mucopolysaccharidoses. As a first step toward the development of an effective gene therapy for mucopolysaccharidosis type IVA (Morquio syndrome), a recombinant retroviral vector, LGSN, derived from the LXSN vector, containing a full-length human wildtype N-acetylgalactosamine-6-sulfate sulfatase (GALNS) cDNA, was produced. Severe Morquio and normal donor fibroblasts were transduced by LGSN. GALNS activity in both Morquio and normal transduced cells was several fold higher than normal values. To measure the variability of GALNS expression among different transduced cells, we transduced normal and Morquio lymphoblastoid B cells and PBLs, human keratinocytes, murine myoblasts C2C12, and rabbit synoviocytes HIG-82 with LGSN. In all cases, an increase of GALNS activity after transduction was measured. In Morquio cells co-cultivated with enzyme-deficient transduced cells, we demonstrated enzyme uptake and persistence of GALNS activity above normal levels for up to 6 days. The uptake was mannose-6-phosphate dependent. Furthermore, we achieved clear evidence that LGSN transduction of Morquio fibroblasts led to correction of the metabolic defect. These results provide the first evidence that GALNS may be delivered either locally or systematically by various cells in an ex vivo gene therapy of MPS IVA.

Metabolic correction in oligodendrocytes derived from metachromatic leukodystrophy mouse model by using encapsulated recombinant myoblasts

In an effort to develop an encapsulated cell-based system to deliver arylsulfatase A (ARSA) to the central nervous system of metachromatic leukodystrophy (MLD) patients, we engineered C2C12 mouse myoblasts with a retroviral vector containing a full-length human ARSA cDNA and evaluated the efficacy of the recombinant secreted enzyme to revert the MLD phenotype in oligodendrocytes (OL) of the As2-/- mouse model. After transduction, C2C12 cells showed a fifteen-fold increase in intracellular ARSA activity and five-fold increase in ARSA secretion. The secreted hARSA collected from transduced cells encapsulated in polyether-sulfone polymer, was taken up by enzyme-deficient OL derived from MLD mice and normally sorted to the lysosomal compartment, where transferred enzyme reached 80% of physiological levels, restoring the metabolism of sulfatide. To evaluate whether secreted enzyme could restore metabolic function in the brain, encapsulated cells and secreted ARSA were shown to be stable in CSF in vitro. Further, to test cell viability and enzyme release in vivo, encapsulated cells were implanted subcutaneously on the dorsal flank of DBA/2J mice. One month later, all retrieved implants released hARSA at rates similar to unencapsulated cells and contained well preserved myoblasts, demonstrating that encapsulation maintains differentiation of C2C12 cells, stable transgene expression and long-term cell viability in vivo. Thus, these results show the promising potential of developing an ARSA delivery system to the CNS based on the use of a polymer-encapsulated transduced xenogenic cell line for gene therapy of MLD.

Detection of PLGA-based nanoparticles at a single-cell level by synchrotron radiation FTIR spectromicroscopy and correlation with X-ray fluorescence microscopy

Poly-lactide-co-glycolide (PLGA) is one of the few polymers approved by the US Food and Drug Administration as a carrier for drug administration in humans; therefore, it is one of the most used materials in the formulation of polymeric nanoparticles (NPs) for therapeutic purposes. Because the cellular uptake of polymeric NPs is a hot topic in the nanomedicine field, the development of techniques able to ensure incontrovertible evidence of the presence of NPs in the cells plays a key role in gaining understanding of their therapeutic potential. On the strength of this premise, this article aims to evaluate the application of synchrotron radiation-based Fourier transform infrared spectroscopy (SR-FTIR) spectromicroscopy and SR X-ray fluorescence (SR-XRF) microscopy in the study of the in vitro interaction of PLGA NPs with cells. To reach this goal, we used PLGA NPs, sized around 200 nm and loaded with superparamagnetic iron oxide NPs (PLGA-IO-NPs; Fe3O4; size, 10–15 nm). After exposing human mesothelial (MeT5A) cells to PLGA-IO-NPs (0.1 mg/mL), the cells were analyzed after fixation both by SR-FTIR spectromicroscopy and SR-XRF microscopy setups. SR-FTIR-SM enabled the detection of PLGA NPs at single-cell level, allowing polymer detection inside the biological matrix by the characteristic band in the 1,700–2,000 cm−1 region. The precise PLGA IR-signature (1,750 cm−1 centered pick) also was clearly evident within an area of high amide density. SR-XRF microscopy performed on the same cells investigated under SR-FTIR microscopy allowed us to put in evidence the Fe presence in the cells and to emphasize the intracellular localization of the PLGA-IO-NPs. These findings suggest that SR-FTIR and SR-XRF techniques could be two valuable tools to follow the PLGA NPs’ fate in in vitro studies on cell cultures.

Synthesis of Lipophilic Core–Shell Fe3O4@SiO2@Au Nanoparticles and Polymeric Entrapment into Nanomicelles: A Novel Nanosystem for in Vivo Active Targeting and Magnetic Resonance–Photoacoustic Dual Imaging

In this work, iron/silica/gold core-shell nanoparticles (Fe3O4@SiO2@Au NPs) characterized by magnetic and optical properties have been synthesized to obtain a promising theranostic platform. To improve their biocompatibility, the obtained multilayer nanoparticles have been entrapped in polymeric micelles, decorated with folic acid moieties, and tested in vivo for photoacoustic and magnetic resonance imaging detection of ovarian cancer.