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Hypothesis Solid lipid nanoparticles (SLNs), co-encapsulating superparamagnetic iron oxide nanoparticles and sorafenib, have been exploited for magnetic-guided drug delivery to the liver. Two different magnetic configurations, both comprising two small magnets, were under-skin implanted to investigate the effect of the magnetic field topology on the magnetic SLNP accumulation in liver tissues. A preliminary simulation analysis was performed to predict the magnetic field topography for each tested configuration. Experiments SLNs were prepared using a hot homogenization approach and characterized using complementary techniques. Their in vitro biological behavior was assessed in HepG-2 liver cancer cells; wild-type mice were used for the in vivo study. The magnet configuration that resulted in a higher magnetic targeting efficiency was investigated by evaluating the iron content in homogenated murine liver tissues. Findings SLNs, characterized by an average size smaller than 200 nm, retained their superparamagnetic behavior and relevant molecular resonance imaging properties as negative contrast agents. The evaluation of iron accumulation in the liver tissues was consistent with the magnetic induction profile of each magnet configuration, concurring with the results predicted by simulation analysis and obtained by measurements in living mice.

The solubility of bortezomib, a boronic acid, in water and normal saline is often misquoted in the literature. Here we confirm that bortezomib equilibrium solubility in water and normal saline is 0.59 ± 0.07 and 0.52 ± 0.11 mg/mL, respectively. The aqueous solubility is significantly enhanced, 1.92 ± 0.14 and 3.40 ± 0.21 mg/mL, respectively, in the presence of 55 mM and 137 mM D-mannitol in normal saline, as in the commercial formulation, Velcade®, after reconstitution. This is due to reversible ester formation between bortezomib and D-mannitol. Based on the pH-solubility profile curve for bortezomib in the absence of added D-mannitol, bortezomib's pKa value is estimated to be 8.8 ± 0.2. Boric acid, glycine and a combination of the two, used in an alternative formulation to that of Velcade® do not enhance the equilibrium aqueous solubility of bortezomib.

A series of compounds generated by ring expansion/opening and molecular elongation/simplification of the 1,3-dioxolane scaffold were prepared and tested for binding affinity at 5-HT1AR and α1 adrenoceptors. The compounds with greater affinity were selected for further functional studies. N-((2,2-diphenyl-1,3-dioxan-5-yl)methyl)-2-(2-methoxyphenoxy)ethan-1-ammonium hydrogen oxalate (12) emerged as highly potent full agonist at the 5-HT1AR (pKi 5-HT1A = 8.8; pD2 = 9.22, %Emax = 92). The pharmacokinetic data in rats showed that the orally administered 12 has a high biodistribution in the brain compartment. Thus, 12 was further investigated in-vivo, showing an anxiolytic and antidepressant effect. Moreover, in the formalin test, 12 was able to decrease the late response to the noxious stimulus, indicating a potential use in the treatment of chronic pain.

Herein we envisaged the possibility of exploiting alkyl nitrates as precursors of alcohol-bearing dual inhibitors targeting acetylcholinesterase (AChE) and monoamine oxidase B (MAO B), key enzymes in neurodegenerative syndromes such as Alzheimer's disease (AD), through biotransformation unmasking an alcoholic function upon nitric oxide (NO) release. The cooperation to neuroprotection of low fluxes of NO and target enzymes' inhibition by the alcohol metabolites might return a multitargeting effect. The in vitro screening towards ChEs and MAOs of a collection of 21 primary alcohols disclosed a subset of dual inhibitors, among which three diverse chemotypes were selected to study the corresponding nitrates. Nitrate 14 proved to be a brain permeant, potent AChE-MAO B inhibitor by itself. Moreover, it protected human SH-SY5Y lines against rotenone and hydrogen peroxide with a poor inherent cytotoxicity and showed a slow conversion profile to its alcohol metabolite 9d that still behaved as bimodal and neuroprotective molecule.

A new model of solar reactor based on a double-loop fluidized bed involving CeO2 nanoparticles and two gas streams, N2 and CO2, for efficient thermochemical fuel production, is presented. The fluidized bed reactors are commonly used to carry out a variety of chemical reactions, due to solid granular materials, which play the fundamental role of catalyst. In the system under investigation, the overall reaction CO2→CO+1⁄2O2 is achieved, by means of a thermochemical two-step cycle, based on CeO2 nanoparticles. The first step (CeO2 thermal reduction) has been implemented with a solar-driven endothermic dissociation of the metal oxide to lower- valence metal-oxide. The second step (CO2 splitting) has been carried out with an exothermic oxidation of the reduced metal-oxide, which is produced in the first step, to form CO. The use of nanoparticles as catalyst allows maximizing the surface area of reaction, and at the same time, the reactor based on double-loop fluidized bed allows continuous operation, without alternating flows of inert sweep gas and CO2. The thermodynamic analysis of the system under investigation showed a calculated maximum ideal efficiency of about 63%.

In this paper a critical investigation of layering phenomenon has been carried out, by means of experimental and numerical analyses, to explain the differences in thermal conductivity between nanofluids based on metal (Cu) and metal oxide (CuO) nanoparticles. Particularly, molecular dynamics simulations have been developed to investigate the adsorption of water molecules surrounding Cu and CuO nanoparticles of various sizes. Furthermore, different volume concentrations of nanoparticles in water have been analyzed. The numerical results revealed two shell-like formations of water molecules (layers) close to the Cu nanoparticle surface, differently from CuO nanoparticle, where no significant layering phenomenon has been observed. This result can explain the higher thermal conductivity of Cu-based nanofluid with respect to CuO-based one, which has been experimentally measured. The numerical and experimental results lead to the conclusion that layers of ordered water molecules surrounding metal nanoparticles play an important role in explaining experimental data of nanofluid thermal conductivity.

The Pt(IV) prodrug of kiteplatin, cis-trans-cis-[PtCl2(RS-Ibuprofen-H)2(cis-1,4-DACH)], having in the axial positions two molecules of Ibuprofen, has been synthesised, characterized and tested in vitro. The aim was to potentiate the cytotoxic effect of kiteplatin with the anti-inflammatory activity of Ibuprofen. The reduction potential of the conjugate resulted comparable to those of other reported Pt(IV) carboxylate complexes, ensuring in vivo stability in blood during transport and intracellular reduction with release of the active species. The cytotoxic activity of the complex resulted remarkably potentiated reaching nanomolar activity. It is possible that the coordinated Ibuprofen molecules promote the transport and the accumulation of the complex in tumor cells by increasing its lipophilicity. In addition to the increased uptake, Ibuprofen could also exert an anti-inflammatory action mediated by inhibition of the COX enzymes which are overexpressed in tumors. The use of a conjugate with Pt-bound Ibuprofen can ensure similar cellular uptake and biodistribution for both the anti-inflammatory and the cytotoxic drugs in the exact ratio of 2:1.

Solid lipid nanoparticles (SLNs) can combine the advantages of different colloidal carriers and prevent some of their disadvantages. The production of nanoparticles by means of microfluidics represents a successful platform for industrial scale-up of nanoparticle manufacture in a reproducible way. The realisation of a microfluidic technique to obtain SLNs in a continuous and reproducible manner encouraged us to create surface functionalised SLNs for targeted drug release using the same procedure. A tumor homing peptide, iRGD, owning a cryptic C-end Rule (CendR) motif is responsible for neuropilin-1 (NRP-1) binding and for triggering extravasation and tumor penetration of the peptide. In this study, the Paclitaxel loaded-SLNs produced by microfluidics were functionalized with the iRGD peptide. The SLNs proved to be stable in aqueous medium andwere characterized by a Z-average under 150 nm, a polydispersity index below 0.2, a zeta-potential between -20 and -35 mV and a drug encapsulation efficiency around 40%. Moreover, in vitro cytotoxic effects and cellular uptake have been assessed using 2D and 3D tumour models of U87 glioblastoma cell lines. Overall, these results demonstrate that the surface functionalization of SLNs with iRGD allow better cellular uptake and cytotoxicity ability.

A promising new generation of solar thermal collector able to enhance the thermal efficiency is the DASC (Direct Absorber Solar Collector). In this paper we report optical absorption measurements performed on several water-based nanofluids (Al2O3, CuO, TiO2, ZnO, CeO2, and Fe2O3) as a function of nanoparticles concentration. These measurements are of fundamental importance to assess the possibility to use the above mentioned metal-oxide nanoparticles in liquid-based nanofluids for direct absorption low temperature flat panel solar collector. The obtained results show different optical behaviors of the nanofluids depending on nanoparticles material and concentration. In all measurements the transmittance rises passing from visible to infrared region and in some cases, when the nanoparticles concentration is too low, the extinction distance grows up to values larger than the typical diameter of a solar receiver.

Reported here is the synthesis and biological evaluation of the asialoglycoprotein receptor (ASGP-R) targeted fourth generation poliamidoamine dendrimer (G(4)-PAMAM) loaded with sorafenib. The ASGP-R targeted dendrimer was obtained by conjugation of Lactobionic acid (La) to the G(4)-PAMAM dendrimer, followed by acetylation (Ac) of the free amino groups in order to reduce the non-specific interactions with the cell membrane. Moreover, by additionally grafting fluorescein (FITC), it was easy to characterize the internalization pathway and the intracellular fate of the targeted dendrimer Ac-La-G(4)-PAMAM-FITC. In vitro experiments performed on HepG-2 and HLE cell lines, allowed to study the ability of the dendrimers to affect the cell vitality. Confocal microscopy and cytofluorimetric analysis confirmed higher binding and uptake ability of the Ac-La-G(4)-PAMAM-FITC dendrimer in well differentiated and ASGP-R expressing human liver cancer cell line HepG-2 compared non-expressing HLE cells. Ac-La-G(4)-PAMAM-FITC dendrimer loaded with sorafenib was stable and showed sustained sorafenib release. As evidenced by the cytotoxicity studies, sorafenib included in the dendrimer maintained its effectiveness, and was able to produce a longer lasting effect over the time compared to molar equivalent doses of free sorafenib. This new targeted dendrimer appears to be a suitable carrier for the delivery of sorafenib to liver cancer cells expressing ASGP-R.

Since the discovery of antitumor activity of cisplatin in the late 60’s, the development of metal-based drugs has had an enormous burst with the aim of to increase the anti-cancer activity, lower side effects and promote specific accumulation to the target cells or organs. Indeed, a new discipline, called medicinal inorganic chemistry, has born where metal ions play a fundamental role. This review provides a comprehensive overview of various metal complexes targeting the Translocator Protein (TSPO), an 18 kDa high affinity cholesterol- and drug-binding protein found mostly in the outer mitochondrial membrane and formerly known as peripheral-type benzodiazepine receptor. TSPO distribution in healthy tissues varies considerably; in contrast, aberrant expression of TSPO has been noted in several pathological specimens including neurodegenerative diseases and cancers. These findings suggest that TSPO-targeted therapy and imaging may have utility in the treatment and visualization of diseases expressing high density of TSPO. In this review, potential applications in cancer diagnosis and therapy will be discussed.

Background Organic cation transporters transfer solutes with a positive charge across the plasma membrane. The novel organic cation transporter 1 (OCTN1) and 2 (OCTN2) transport ergothioneine and carnitine, respectively. Mutations in the SLC22A5 gene encoding OCTN2 cause primary carnitine deficiency, a recessive disorders resulting in low carnitine levels and defective fatty acid oxidation. Variations in the SLC22A4 gene encoding OCTN1 are associated with rheumatoid arthritis and Crohn disease. Methods Here we evaluate the functional properties of the OCTN1 transporter using chimeric transporters constructed by fusing different portion of the OCTN1 and OCTN2 cDNAs. Their relative abundance and subcellular distribution was evaluated through western blot analysis and confocal microscopy. Results Substitutions of the C-terminal portion of OCTN1 with the correspondent residues of OCTN2 generated chimeric OCTN transporters more active than wild-type OCTN1 in transporting ergothioneine. Additional single amino acid substitutions introduced in chimeric OCTN transporters further increased ergothioneine transport activity. Kinetic analysis indicated that increased transport activity was due to an increased V max , with modest changes in K m toward ergothioneine. Conclusions Our results indicate that the OCTN1 transporter is tolerant to extensive amino acid substitutions. This is in sharp contrast to the OCTN2 carnitine transporter that has been selected for high functional activity through evolution, with almost all substitutions reducing carnitine transport activity. General significance The widespread tolerance of OCTN1 to amino acid substitutions suggests that the corresponding SLC22A4 gene may have derived from a recent duplication of the SLC22A5 gene and might not yet have a defined physiological role.

The mitochondrial citrate–malate exchanger (CIC), a known target of acetylation, is up-regulated in activated immune cells and plays a key role in the production of inflammatory mediators. However, the role of acetylation in CIC activity is elusive. We show that CIC is acetylated in activated primary human macrophages and U937 cells and the level of acetylation is higher in glucose-deprived compared to normal glucose medium. Acetylation enhances CIC transport activity, leading to a higher citrate efflux from mitochondria in exchange with malate. Cytosolic citrate levels do not increase upon activation of cells grown in deprived compared to normal glucose media, indicating that citrate, transported from mitochondria at higher rates from acetylated CIC, is consumed at higher rates. Malate levels in the cytosol are lower in activated cells grown in glucose-deprived compared to normal glucose medium, indicating that this TCA intermediate is rapidly recycled back into the cytosol where it is used by the malic enzyme. Additionally, in activated cells CIC inhibition increases the NADP+/NADPH ratio in glucose-deprived cells; this ratio is unchanged in glucose-rich grown cells due to the activity of the pentose phosphate pathway. Consistently, the NADPH-producing isocitrate dehydrogenase level is higher in activated glucose-deprived as compared to glucose rich cells. These results demonstrate that, in the absence of glucose, activated macrophages increase CIC acetylation to enhance citrate efflux from mitochondria not only to produce inflammatory mediators but also to meet the NADPH demand through the actions of isocitrate dehydrogenase and malic enzyme.

Anigopreissin A belongs to stilbene di- and oligomeric forms containing a benzofuran ring system whose biological activity is unknown. Recently, a completely protected Anigopreissin A - Permethylated Anigopreissin A - has been synthesized. We use MTT bioassay to assess Permethylated Anigopreissin A cytotoxicity in different human cell lines. Furthermore, fluorescence microscopy, caspase activity, real-time PCR and Western-blot methods are employed to evaluate apoptotic cell death pathway in liver cancer cells. Permethylated Anigopreissin A kills different types of human cancer cells but does not affect non-tumorigenic cells. The Permethylated Anigopreissin A concentration that causes 50% inhibition of liver tumor cells is 0.24μM. Hepatoma cells treated with Permethylated Anigopreissin A arrest their cell cycle in G1 phase. We also demonstrate that Permethylated Anigopreissin A-triggered cell death occurs by apoptosis. Decrease of the BCL2 expression levels, loss of the mitochondrial membrane potential, release of cytochrome c and increase of caspase 9 activity highlight a key role for mitochondria in Permethylated Anigopreissin A-induced apoptosis. Our study shows that Permethylated Anigopreissin A kills liver cancer cells through intrinsic apoptotic pathway.

Curcumin (CR) is a natural polyphenol with antioxidative, anti-inflammatory, and anticancer properties but its therapeutic potential is substantially hindered by the rather low-water solubility and bioavailability. Thus, in this work, a new soluble inclusion complex of CR with sulfobutylether-β-cyclodextrin (SBE-β-CD) was prepared in solution and at the solid state using different preparation techniques and characterized by Fourier transform infrared, nuclear magnetic resonance, differential scanning calorimetry, scanning electron microscopy, phase solubility studies, and Job's plot method. Results clearly indicate that CR reacts with SBE-β-CD to form a host-guest complex with an apparent formation constant of 1455 M(-1) . Moreover, SBE-β-CD strongly increases water solubility of CR (from 0.56 to 102.78 μg/mL, at 25°C), and lyophilization method seems to be the best preparation technique to obtain the complex at the solid state. Finally, an in vitro test on a human hepatic cancer cell line (HepG-2) shows that complexation positively influences CR anticancer and antioxidant activity.

Myocyte enhancer factor 2C (MEF2C) belongs to the MEF2 transcription factors. All products of MEF2 genes have a common amino-terminal DNA binding and dimerization domain. All four vertebrate MEF2 gene transcripts are also alternatively spliced. In the present study we identify two novel MEF2C splice variants, named VP and VP2. These variants are generated by the skipping of exon α. The identified α- variants are ubiquitously expressed, although at very low levels compared to the α+ variants. The existence of MEF2C α- variants gave us the opportunity to study for the first time the function of exon α. Transactivation experiments show that the presence of exon α induces a reduction of transcription levels. Moreover, α- variants are significantly expressed during neuronal cell differentiation, indicating a putative role of these variants in development.

Mitochondria represent an attractive subcellular target due to its function particularly important for oxidative damage, calcium metabolism and apoptosis. However, the concept of mitochondrial targeting has been a neglected area so far. The translocator protein (TSPO) represents an interesting subcellular target not only to image disease states overexpressing this protein, but also for a selective mitochondrial drug targeting. Recently, we have delivered in vitro and in vivo small molecule imaging agents into cells overexpressing TSPO by using a family of high-affinity conjugable ligands characterized by 2-phenyl-imidazo[1,2-a]pyridine acetamide structure. As an extension, in the present work we studied the possibility to target and image TSPO with dendrimers. These nano-platforms have unique features, in fact, are prepared with a level of control not reachable with most linear polymers, leading to nearly monodisperse, globular macromolecules with a large number of peripheral groups. As a consequence, they are an ideal delivery vehicle candidate for explicit study of the effects of polymer size, charge, composition, and architecture on biologically relevant properties such as lipid bilayer interactions, cytotoxicity, cellular internalization, and subcellular compartments and organelles interactions. Here, we present the synthesis, characterization, cellular internalization, and mitochondria labeling of a TSPO targeted fourth generation [G(4)-PAMAM] dendrimer nanoparticle labeled with the organic fluorescent dye fluorescein. We comprehensively studied the cellular uptake behavior of these dendrimers, into glioma C6 cell line, under the influence of various endocytosis inhibitors. We found that TSPO targeted-G(4)-PAMAM-FITC dendrimer is quickly taken up by these cells by endocytosis pathways, and moreover specifically targets the mitochondria as evidenced from subcellular fractionation experiments and co-localization studies performed with CAT (Confocal-AFM-TIRF) microscopy.

Disorders of the carnitine cycle and of the beta oxidation spiral impair the ability to obtain energy from fats at time of fasting and stress. This can result in hypoketotic hypoglycemia, cardiomyopathy, cardiac arrhythmia and other chronic medical problems. The in vitro study of fibroblasts from patients with these conditions is impaired by their limited oxidative capacity. Here we evaluate the capacity of valinomycin, a potassium ionophore that increases mitochondrial respiration, to increase the oxidation of fatty acids in cells from patients with inherited fatty acid oxidation defects. The addition of valinomycin to fibroblasts decreased the accumulation of the lipophilic cation tetraphenylphosphonium (TPP(+)) at low concentrations due to the dissipation of the mitochondrial membrane potential. At higher doses, valinomycin increased TPP(+) accumulation due to the increased potassium permeability of the plasma membrane and subsequent cellular hyperpolarization. The incubation of normal fibroblasts with valinomycin increased [(14)C]-palmitate oxidation (measured as [(14)C]O2 release) in a dose-dependent manner. By contrast, valinomycin failed to increase palmitate oxidation in fibroblasts from patients with very long chain acyl CoA dehydrogenase (VLCAD) deficiency. This was not observed in fibroblasts from patients heterozygous for this condition. These results indicate that valinomycin can increase fatty acid oxidation in normal fibroblasts and could be useful to differentiate heterozygotes from patients affected with VLCAD deficiency.

The citrate carrier (CIC), a nuclear-encoded protein located in the mitochondrial inner membrane, plays an important metabolic role in the transport of acetyl-CoA from the mitochondrion to the cytosol in the form of citrate for fatty acid and cholesterol synthesis. Citrate has been reported to be essential for fibroblast differentiation into fat cells. Because peroxisome proliferator-activated receptor-gamma (PPARγ) is known to be one of the master regulators of adipogenesis, we aimed to study the regulation of CIC by the PPARγ ligand rosiglitazone (BRL) in 3T3-L1 fibroblasts and in adipocytes. We demonstrated that BRL up-regulated CIC mRNA and protein levels in fibroblasts, while it did not elicit any effects in mature adipocytes. The enhancement of CIC levels upon BRL treatment was reversed using the PPARγ antagonist GW9662, addressing how this effect was mediated by PPARγ. Functional experiments using a reporter gene containing rat CIC promoter showed that BRL enhanced CIC promoter activity. Mutagenesis studies, electrophoretic-mobility-shift assay and chromatin-immunoprecipitation analysis revealed that upon BRL treatment, PPARγ and Sp1 are recruited on the Sp1-containing region within the CIC promoter, leading to an increase in CIC expression. In addition, mithramycin, a specific inhibitor for Sp1-DNA binding activity, abolished the PPARγ-mediated up-regulation of CIC in fibroblasts. The stimulatory effects of BRL disappeared in mature adipocytes in which PPARγ/Sp1 complex recruited SMRT corepressor to the Sp1 site of the CIC promoter. Taken together, our results contribute to clarify the molecular mechanisms by which PPARγ regulates CIC expression during the differentiation stages of fibroblasts into mature adipocytes.

The carnitine/acylcarnitine carrier (CAC) is a transport protein of the inner mitochondrial membrane that belongs to the mitochondrial carrier protein family. In its cytosolic conformation the carrier consists of a bundle of six transmembrane α-helices, which delimit a water filled cavity opened towards the cytosol and closed towards the matrix by a network of interacting charged residues. Most of the functional data on this transporter come from studies performed with the protein purified from rat liver mitochondria or recombinant proteins from different sources incorporated into phospholipid vesicles (liposomes). The carnitine/acylcarnitine carrier transports carnitine and acylcarnitines with acyl chains of various lengths from 2 to 18 carbon atoms. The mammalian transporter exhibits higher affinity for acylcarnitines with longer carbon chains. The functional data indicate that CAC plays the important function of catalyzing transport of acylcarnitines into the mitochondria in exchange for intramitochondrial free carnitine. This results in net transport of fatty acyl units into the mitochondrial matrix where they are oxidized by the β-oxidation enzymes. The essential role of the transporter in cell metabolism is demonstrated by the fact that alterations of the human gene SLC25A20 coding for CAC are associated with a severe disease known as carnitine carrier deficiency. This autosomal recessive disorder is characterized by life-threatening episodes of coma induced by fasting, cardiomyopathy, liver dysfunction, muscle weakness, respiratory distress and seizures. Until now 35 different mutations of CAC gene have been identified in carnitine carrier deficient patients. Some missense mutations concern residues of the signature motif present in all mitochondrial carriers. Diagnosis of carnitine carrier deficiency requires biochemical and genetic tests; treatment is essentially limited to important dietetic measures. Recently, a pharmacological approach based on the use of statins and/or fibrates for the treatment of CAC-deficient patients with mild phenotype has been proposed.

In the last few years, we have been functionally characterizing the promoter of the human mitochondrial citrate carrier (CIC). In this study we show that CIC silencer activity extends over 26 bp (-595/-569), which specifically bind a protein present in HepG2 cell nuclear extracts. This transcription factor was purified by DNA affinity and identified as ZNF224. Overexpression of ZNF224 decreases LUC transgene activity in cells transfected with a construct containing the CIC silencer region, whereas ZNF224 silencing activates reporter transcription in cells transfected with the same construct. Moreover, overexpression and silencing of ZNF224 diminishes and enhances, respectively, CIC transcript and protein levels. Finally, ZNF224 is abundantly expressed in fetal tissues contrary to CIC. It is suggested that CIC transcriptional repression by ZNF224 explains, at least in part, the low expression of CIC in fetal tissues in which fatty acid synthesis is low.