Leopoldo Sitia

Blood protein coating of gold nanoparticles as potential tool for organ targeting.

Nanoparticles (NP) and nanoparticulated drug delivery promise to be the breakthrough for therapy in medicine but raise concerns in terms of nanotoxicity. We present quantitative murine biokinetics assays using polyelectrolyte-multilayer-coated gold NP (AuNP, core diameter 15 and 80 nm; (198)Au radio-labeled). Those were stably conjugated either with human serum albumin (alb-AuNP) or apolipoprotein E (apoE-AuNP), prior to intravenous injection. We compare the biokinetics of protein-AuNP-conjugates with citrate-stabilized AuNP (cit-AuNP). Biokinetics was complemented with histology in organs with high AuNP content using 15 nm double fluorescently-labeled alb-AuNP-conjugates. Protein conjugation massively reduced liver retention (alb-AuNP: 52%, apoE-AuNP: 72%, cit-AuNP: >95%, at 19 h and 48 h) when compared to cit-AuNP. The protein conjugates were retained in lungs (alb-AuNP (18%) and spleen (alb-AuNP (16%), apoE-AuNP (21%) at 19 h. Alb-AuNP show significantly increased fractions in lungs (factors: 60 (30 min); 111 (19 h); 235 (48 h) and brain (factors: 70 (30 min); 90 (19 h); >200 (48 h) compared to cit-AuNP (control) - or even to apoE-AuNP. The influence of protein conjugation on the biodistribution disappears for 80 nm AuNP comparing to control. Histologically, the 15 nm alb-AuNP are mainly located in the endothelium of brain, lungs, liver and kidneys after 30 min, while at 19 h they moved deeper into the parenchyma e.g. in hippocampus. Our study clearly suggests that stable conjugation of AuNP with albumin and apoE prior to intravenous administration increases specificity and efficiency of NP in diseased target-organs thus suggesting a potential role in nanomedicine and nanopharmacology.

Biocompatible fluorescent nanoparticles for in vivo stem cell tracking

Efficient application of stem cells to the treatment of neurodegenerative diseases requires safe cell tracking to follow stem cell fate over time in the host environment after transplantation. In this work, for the first time, fluorescent and biocompatible methyl methacrylate (MMA)-based nanoparticles (fluoNPs) were synthesized through a free-radical co-polymerization process with a fluorescent macromonomer obtained by linking Rhodamine B and hydroxyethyl methacrylate. We demonstrate that the fluoNPs produced by polymerization of MMA-Rhodamine complexes (1) were efficient for the labeling and tracking of multipotent human amniotic fluid cells (hAFCs); (2) did not alter the main biological features of hAFCs (such as viability, cell growth and metabolic activity); (3) enabled us to determine the longitudinal bio-distribution of hAFCs in different brain areas after graft in the brain ventricles of healthy mice by a direct fluorescence-based technique. The reliability of our approach was furthermore confirmed by magnetic resonance imaging analyses, carried out by incubating hAFCs with both superparamagnetic iron oxide nanoparticles and fluoNPs. Our data suggest that these finely tunable and biocompatible fluoNPs can be exploited for the longitudinal tracking of stem cells.

Genetic Analysis Reveals a Longevity-Associated Protein Modulating Endothelial Function and Angiogenesis

RATIONALE Long living individuals show delay of aging, which is characterized by the progressive loss of cardiovascular homeostasis, along with reduced endothelial nitric oxide synthase activity, endothelial dysfunction, and impairment of tissue repair after ischemic injury. OBJECTIVE Exploit genetic analysis of long living individuals to reveal master molecular regulators of physiological aging and new targets for treatment of cardiovascular disease. METHODS AND RESULTS We show that the polymorphic variant rs2070325 (Ile229Val) in bactericidal/permeability-increasing fold-containing-family-B-member-4 (BPIFB4) associates with exceptional longevity, under a recessive genetic model, in 3 independent populations. Moreover, the expression of BPIFB4 is instrumental to maintenance of cellular and vascular homeostasis through regulation of protein synthesis. BPIFB4 phosphorylation/activation by protein-kinase-R-like endoplasmic reticulum kinase induces its complexing with 14-3-3 and heat shock protein 90, which is facilitated by the longevity-associated variant. In isolated vessels, BPIFB4 is upregulated by mechanical stress, and its knock-down inhibits endothelium-dependent vasorelaxation. In hypertensive rats and old mice, gene transfer of longevity-associated variant-BPIFB4 restores endothelial nitric oxide synthase signaling, rescues endothelial dysfunction, and reduces blood pressure levels. Furthermore, BPIFB4 is implicated in vascular repair. BPIFB4 is abundantly expressed in circulating CD34(+) cells of long living individuals, and its knock-down in endothelial progenitor cells precludes their capacity to migrate toward the chemoattractant SDF-1. In a murine model of peripheral ischemia, systemic gene therapy with longevity-associated variant-BPIFB4 promotes the recruitment of hematopoietic stem cells, reparative vascularization, and reperfusion of the ischemic muscle. CONCLUSIONS Longevity-associated variant-BPIFB4 may represent a novel therapeutic tool to fight endothelial dysfunction and promote vascular reparative processes.

In Vivo Fate of Avidin-Nucleic Acid Nanoassemblies as Multifunctional Diagnostic Tools

This study describes the formulation optimization and body-cell distribution and clearance in mice of a dually fluorescent biodegradable poly avidin nanoassembly based on the novel Avidin-Nucleic-Acid-Nano-ASsembly (ANANAS) platform as a potential advancement of classic avidin/biotin-based targeted delivery. The nanoformulation circulates freely in the bloodstream; it is slowly captured by filter organs; it is efficiently cleared within 24-48 h, and it is poorly immunogenic. The system displays more favorable properties than its parent monomeric avidin and it is a promising tool for diagnostic purposes for future translational aims, for which free circulation in the bloodstream, safety, multifunctionality and high composition definition are all necessary requirements. In addition, the assembly shows a time-dependent cell penetration capability, suggesting it may also function as a NP-dependent drug delivery tool. The ease of preparation together with the possibility to fine-tune the surface composition makes it also an ideal candidate to understand if and how nanoparticle composition affects its localization.

Longitudinal Tracking of Human Fetal Cells Labeled with Super Paramagnetic Iron Oxide Nanoparticles in the Brain of Mice with Motor Neuron Disease

Stem Cell (SC) therapy is one of the most promising approaches for the treatment of Amyotrophic Lateral Sclerosis (ALS). Here we employed Super Paramagnetic Iron Oxide nanoparticles (SPIOn) and Hoechst 33258 to track human Amniotic Fluid Cells (hAFCs) after transplantation in the lateral ventricles of wobbler (a murine model of ALS) and healthy mice. By in vitro, in vivo and ex vivo approaches we found that: 1) the main physical parameters of SPIOn were maintained over time; 2) hAFCs efficiently internalized SPIOn into the cytoplasm while Hoechst 33258 labeled nuclei; 3) SPIOn internalization did not alter survival, cell cycle, proliferation, metabolism and phenotype of hAFCs; 4) after transplantation hAFCs rapidly spread to the whole ventricular system, but did not migrate into the brain parenchyma; 5) hAFCs survived for a long time in the ventricles of both wobbler and healthy mice; 6) the transplantation of double-labeled hAFCs did not influence mice survival.

A biodistribution study of PEGylated PCL-based nanoparticles in C57BL/6 mice bearing B16/F10 melanoma

One of the major drawbacks that limits the clinical application of nanoparticles is the lack of preliminary investigations related to their biocompatibility, biodegradability and biodistribution. In this work, biodegradable PEGylated polymer nanoparticles (NPs) have been synthesized by using macromonomers based on poly(ε-caprolaconte) oligomers. More in detail, NPs have been produced by adopting a surfactant-free semibatch emulsion polymerization process using PEG chains as a stabilizing agent. The NPs were also labeled with rhodamine B covalently bound to the NPs to quantitatively study their biodistribution in vivo. NPs were investigated in both in vitro and in vivo preclinical systems to study their biodistribution in mice bearing B16/F10 melanoma, as well as their biocompatibility and biodegradability. The NP concentration was evaluated in different tissues at several times after intravenous injection. The disappearance of the NPs from the plasma was biphasic, with distribution and elimination half-lives of 30 min and 15 h, respectively. NPs were retained in tumors and in filter organs for a long time, were still detectable after 7 d and maintained a steady concentration in the tumor for 120 h. 48 h after injection, 70 ± 15% of the inoculated NPs were excreted in the feces. The favorable tumor uptake, fast excretion and absence of cytotoxicity foster the further development of produced NPs as drug delivery carriers.

Fate of PLA and PCL-Based Polymeric Nanocarriers in Cellular and Animal Models of Triple-Negative Breast Cancer.

An integrated platform to assess the interaction between nanocarriers and biological matrices has been developed by our group using poly methyl-methacrylate nanoparticles. In this study, we exploited this platform to evaluate the behavior of two biodegradable formulations, poly-ε-caprolactone (PCL3) and poly lactic-acid (PLA8), respectively, in cellular and animal models of triple-negative breast cancer (TNBC). Both NPs shared the main physicochemical parameters (size, shape, ζ-potential) and exclusively differentiated on the material on which they are composed. Our results showed that (1) PLA8 NPs, systemically injected in mice, underwent rapid degradation without penetration into tumors; (2) PLA8 NPs were not internalized in the human TNBC cell line (MDA-MB-231); (3) PCL3 NPs had a longer bioavailability, reached the tumor parenchyma, and efficiently penetrated in MDA-MB-231 cells. Our data highlight the relevance of the material selection to both improve bioavailability and target tropism, and make PCL3 NPs an interesting tool for the development of nanodrugs against TNBC.

Longitudinal tracking of triple labeled umbilical cord derived mesenchymal stromal cells in a mouse model of Amyotrophic Lateral Sclerosis

The translational potential of cell therapy to humans requires a deep knowledge of the interaction between transplanted cells and host tissues. In this study, we evaluate the behavior of umbilical cord mesenchymal stromal cells (UC-MSCs), labeled with fluorescent nanoparticles, transplanted in healthy or early symptomatic transgenic SOD1G93A mice (a murine model of Amyotrophic Lateral Sclerosis). The double labeling of cells with nanoparticles and Hoechst-33258 enabled their tracking for a long time in both cells and tissues. Whole-body distribution of UC-MSCs was performed by in-vivo and ex-vivo analyses 1, 7, 21 days after single intravenous or intracerebroventricular administration. By intravenous administration cells were sequestered by the lungs and rapidly cleared by the liver. No difference in biodistribution was found among the two groups. On the other hand, UC-MSCs transplanted in lateral ventricles remained on the choroid plexus for the whole duration of the study even if decreasing in number. Few cells were found in the spinal cord of SOD1G93A mice exclusively. No migration in brain parenchyma was observed. These results suggest that the direct implantation in brain ventricles allows a prolonged permanence of cells close to the damaged areas and makes this method of tracking reliable for future studies of efficacy.

Lipofuscin accumulation and gene expression in different tissues of mnd mice.

Neuronal ceroid lipofuscinoses (NCLs) are a group of lysosomal storage diseases characterized by neurological impairment and blindness. NCLs are almost always due to single mutations in different genes (CLN1–CLN8). Ubiquitous accumulation of undigested material and of a hydrophobic inner mitochondrial membrane protein, the subunit c of mitochondrial ATP synthase, has been described. Although protein mutation(s) in the endoplasmic reticulum–lysosomes axis can modify the trafficking and the recycling of different molecules, one of the upstream targets in these diseases may be represented by the balance of gene expression. To understand if and how neurons modify the levels of important genes during the first phases of the disease, it is important to characterize the mechanisms of neurodegeneration. Due to the impossibility of performing this analysis in humans, alternative models of investigation are required. In this study, a mouse model of human NCL8, the mnd mouse has been employed. The mnd mice recapitulate many clinical and histopathological features described in NCL8 patients. In this study, we found an altered expression of different genes in both central and peripheral organs associated with lipopigment accumulation. This is a preliminary approach, which could also be of interest in providing new diagnostic tools for NCLs.