Synthesis, Characterization, Fluorescence Properties, and DFT Modeling of Difluoroboron Biindolediketonates.

We report a simple and efficient strategy to enhance the fluorescence of biocompatible biindole diketonates (bdks) in the visible spectrum through difluoroboronation (BF2bdks complexes). Emission spectroscopy testifies an increase in the fluorescence quantum yields from a few percent to as much as >0.7. This massive increment is essentially independent of substitutions at the indole (-H, -Cl, and -OCH3) and corresponds to a significant stabilization of the excited state with respect to non-radiative decay mechanisms: the non-radiative decay rates are reduced by as much as an order of magnitude, from 109 s-1 to 108 s-1, upon difluoroboronation. The stabilization of the excited state is large enough to enable sizeable 1O2 photosensitized production. Different time-dependent (TD) density functional theory (DFT) methods were assessed in their ability to model the electronic properties of the compounds, with TD-B3LYP-D3 providing the most accurate excitation energies. The calculations associate the first active optical transition in both the bdks and BF2bdks electronic spectra to the S0 → S1 transition, corresponding to a shift in the electronic density from the indoles to the oxygens or the O-BF2-O unit, respectively.

Mercury Clathration-Driven Phase Transition in a Luminescent Bipyrazolate Metal-Organic Framework: A Multitechnique Investigation.

Mercury is one of the most toxic heavy metals. By virtue of its triple bond, the novel ligand 1,2-bis(1H-pyrazol-4-yl)ethyne (H2BPE) was expressly designed and synthesized to devise metal-organic frameworks (MOFs) exhibiting high chemical affinity for mercury. Two MOFs, Zn(BPE) and Zn(BPE)·nDMF [interpenetrated i-Zn and noninterpenetrated ni-Zn·S, respectively; DMF = dimethylformamide], were isolated as microcrystalline powders. While i-Zn is stable in water for at least 15 days, its suspension in HgCl2 aqueous solutions prompts its conversion into HgCl2@ni-Zn. A multitechnique approach allowed us to shed light onto the observed HgCl2-triggered i-Zn-to-HgCl2@ni-Zn transformation at the molecular level. Density functional theory calculations on model systems suggested that HgCl2 interacts via the mercury atom with the carbon-carbon triple bond exclusively in ni-Zn. Powder X-ray diffraction enabled us to quantify the extent of the i-Zn-to-HgCl2@ni-Zn transition in 100-5000 ppm HgCl2 (aq) solutions, while X-ray fluorescence and inductively coupled plasma-mass spectrometry allowed us to demonstrate that HgCl2 is quantitatively sequestered from the aqueous phase. Irradiating at 365 nm, an intense fluorescence is observed at 470 nm for ni-Zn·S, which is partially quenched for i-Zn. This spectral benchmark was exploited to monitor in real time the i-Zn-to-HgCl2@ni-Zn conversion kinetics at different HgCl2 (aq) concentrations. A sizeable fluorescence increase was observed, within a 1 h time lapse, even at a concentration of 5 ppb. Overall, this comprehensive investigation unraveled an intriguing molecular mechanism, featuring the disaggregation of a water-stable MOF in the presence of HgCl2 and the self-assembly of a different crystalline phase around the pollutant, which is sequestered and simultaneously quantified by means of a luminescence change. Such a case study might open the way to new-conception strategies to achieve real-time sensing of mercury-containing pollutants in wastewaters and, eventually, pursue their straightforward and cost-effective purification.

Asymmetric Phenyl Substitution: An Effective Strategy to Enhance the Photosensitizing Potential of Curcuminoids.

Curcumin has been demonstrated to exhibit photosensitized bactericidal activity. However, the full exploitation of curcumin as a photo-pharmaceutical active principle is hindered by fast deactivation of the excited state through the transfer of the enol proton to the keto oxygen. Introducing an asymmetry in the molecular structure through acting on the phenyl substituents is expected to be a valuable strategy to impair this undesired de-excitation mechanism competing with the therapeutically relevant ones. In this study, two asymmetric curcumin analogs were synthesized and characterized as to their electronic-state transition spectroscopic properties. Fluorescence decay distributions were also reconstructed. Their analysis confirmed the substantial stabilization of the fluorescent state with respect to the parent compound. Nuclear magnetic resonance experiments were performed with the aim of determining the structural features of the keto-enol ring and the strength of the keto-enol hydrogen bond. Electronic structure calculations were also undertaken to elucidate the effects of substitution on the features of the keto-enol semi-aromatic system and the proneness to proton transfer. Finally, their singlet oxygen-generation efficiency was compared to that of curcumin through the 9,10-dimethylanthracene fluorescent assay.

Double-stranded flanking ends affect the folding kinetics and conformational equilibrium of G-quadruplexes forming sequences within the promoter of KIT oncogene.

G-quadruplexes embedded within promoters play a crucial role in regulating the gene expression. KIT is a widely studied oncogene, whose promoter contains three G-quadruplex forming sequences, c-kit1, c-kit2 and c-kit*. For these sequences available studies cover ensemble and single-molecule analyses, although for kit* the latter were limited to a study on a promoter domain comprising all of them. Recently, c-kit2 has been reported to fold according to a multi-step process involving folding intermediates. Here, by exploiting fluorescence resonance energy transfer, both in ensemble and at the single molecule level, we investigated the folding of expressly designed constructs in which, alike in the physiological context, either c-kit2 or c-kit* are flanked by double stranded DNA segments. To assess whether the presence of flanking ends at the borders of the G-quadruplex affects the folding, we studied under the same protocols oligonucleotides corresponding to the minimal G-quadruplex forming sequences. Data suggest that addition of flanking ends results in biasing both the final equilibrium state and the folding kinetics. A previously unconsidered aspect is thereby unravelled, which ought to be taken into account to achieve a deeper insight of the complex relationships underlying the fine tuning of the gene-regulatory properties of these fascinating DNA structures.

Synthesis, Characterization and DNA-Binding Affinity of a New Zinc(II) Bis(5-methoxy-indol-3-yl)propane-1,3-dione Complex.

The novel zinc(II) µ-oxo-bridged-dimeric complex [Zn2(µ-O)2(BMIP)2] (BMIP = 1,3-bis(5-methoxy-1-methyl-1H-indol-3-yl)propane-1,3-dione), 1, was synthetized and fully characterized. The spectral data indicate a zincoxane molecular structure, with the BMIP ligand coordinating in its neutral form via its oxygen atoms. Structural changes in 1 in dimethylsulfoxide (DMSO) were evidenced by means of spectroscopic techniques including infrared absorption and nuclear magnetic resonance, showing DMSO entrance in the coordination sphere of the metal ion. The resulting complex [Zn2(µ-O)2(BMIP)2(DMSO)], 2, readily reacts in the presence of N-methyl-imidazole (NMI), a liquid-phase nucleoside mimic, to form [Zn2(µ-O)2(BMIP)2(NMI)], 3, through DMSO displacement. The three complexes show high thermal stability, demonstrating that 1 has high affinity for hard nucleophiles. Finally, with the aim of probing the suitability of this system as model scaffold for new potential anticancer metallodrugs, the interactions of 1 with calf thymus DNA were investigated in vitro in pseudo-physiological environment through UV-Vis absorption and fluorescence emission spectroscopy, as well as time-resolved fluorescence studies. The latter analyses revealed that [Zn2(µ-O)2(BMIP)2(DMSO)] binds to DNA with high affinity upon DMSO displacement, opening new perspectives for the development of optimized drug substances.

Nanomechanics of G-quadruplexes within the promoter of the KIT oncogene.

G-quadruplexes (G4s) are tetrahelical DNA structures stabilized by four guanines paired via Hoogsteen hydrogen bonds into quartets. While their presence within eukaryotic DNA is known to play a key role in regulatory processes, their functional mechanisms are still under investigation. In the present work, we analysed the nanomechanical properties of three G4s present within the promoter of the KIT proto-oncogene from a single-molecule point of view through the use of magnetic tweezers (MTs). The study of DNA extension fluctuations under negative supercoiling allowed us to identify a characteristic fingerprint of G4 folding. We further analysed the energetic contribution of G4 to the double-strand denaturation process in the presence of negative supercoiling, and we observed a reduction in the energy required for strands separation.

Demonstration of fibrinogen-FcRn binding at acidic pH by means of Fluorescence Correlation Spectroscopy.

The neonatal Fc receptor (FcRn) interacts with IgG and albumin at acidic pH within endosomes, thus protecting these plasma proteins from degradation. Recently, we proposed fibrinogen as a new binding partner of FcRn. This work was aimed at providing a direct demonstration of FcRn-fibrinogen binding at acidic pH by Fluorescence Correlation Spectroscopy. The increase in diffusion time between free and fibrinogen-bound FITC-labelled FcRn was assumed as the binding indicator. We observed that, at acidic pH (pH = 5.3), FcRn diffusion time shifted from ≈730 µs (FITC-labelled FcRn alone) to >1200 µs (FITC-labelled FcRn added with fibrinogen). A similar trend was exhibited by albumin, a known FcRn interactor, while no significant variations in diffusion time were observed upon incubation with catalase as negative control. Our results demonstrate a binding interaction between fibrinogen, one of the most abundant plasma proteins, and FcRn, a receptor involved in the regulation of the levels of IgG and albumin. This interaction is likely responsible for fibrinogen protection from intracellular degradation and recycling in plasma. Fibrinogen is crucial not only in haemostasis but also in acute inflammatory response and in some pathological conditions. The interaction with FcRn can influence not only the levels of fibrinogen in plasma and other tissues, but also the levels of other FcRn binding partners, among which are some plasma proteins of clinical relevance.

ETNK1 mutations induce a mutator phenotype that can be reverted with phosphoethanolamine.

Recurrent somatic mutations in ETNK1 (Ethanolamine-Kinase-1) were identified in several myeloid malignancies and are responsible for a reduced enzymatic activity. Here, we demonstrate in primary leukemic cells and in cell lines that mutated ETNK1 causes a significant increase in mitochondrial activity, ROS production, and Histone H2AX phosphorylation, ultimately driving the increased accumulation of new mutations. We also show that phosphoethanolamine, the metabolic product of ETNK1, negatively controls mitochondrial activity through a direct competition with succinate at mitochondrial complex II. Hence, reduced intracellular phosphoethanolamine causes mitochondria hyperactivation, ROS production, and DNA damage. Treatment with phosphoethanolamine is able to counteract complex II hyperactivation and to restore a normal phenotype.

Assessment of a Silicon-Photomultiplier-Based Platform for the Measurement of Intracellular Calcium Dynamics with Targeted Aequorin.

Ca2+ is among the most important intracellular second messengers participating in a plethora of biological processes, and the measurement of Ca2+ fluctuations is significant in the phenomenology of the underlying processes. Aequorin-based Ca2+ probes represent an invaluable tool for reliable measurement of Ca2+ concentrations and dynamics in different subcellular compartments. However, their use is limited due to the lack on the market of ready-to-use, cost-effective, and portable devices for the detection and readout of the low-intensity bioluminescence signal produced by these probes. Silicon photomultipliers (SiPMs) are rapidly evolving solid-state sensors for low light detection, with single photon sensitivity and photon number resolving capability, featuring low cost, low voltage, and compact format. Thus, they may represent the sensors of choice for the development of such devices and, more in general, of a new generation of multipurpose bioluminescence detectors suitable for cell biology studies. Ideally, a detector customized for these purposes must combine high dynamic range with high fidelity in reconstructing the light intensity signal temporal profile. In this article, the ability to perform aequorin-based intracellular Ca2+ measurements using a multipurpose, low-cost setup exploiting SiPMs as the sensors is demonstrated. SiPMs turn out to assure performances comparable to those exhibited by a custom-designed photomultiplier tube-based aequorinometer. Moreover, the flexibility of SiPM-based devices might pave the way toward routinely and wide scale application of innovative biophysical protocols.

The Clustering of mApoE Anti-Amyloidogenic Peptide on Nanoparticle Surface Does Not Alter Its Performance in Controlling Beta-Amyloid Aggregation.

The deposition of amyloid-ß (Aß) plaques in the brain is a significant pathological signature of Alzheimer's disease, correlating with synaptic dysfunction and neurodegeneration. Several compounds, peptides, or drugs have been designed to redirect or stop Aß aggregation. Among them, the trideca-peptide CWG-LRKLRKRLLR (mApoE), which is derived from the receptor binding sequence of apolipoprotein E, is effectively able to inhibit Aß aggregation and to promote fibril disaggregation. Taking advantage of Atomic Force Microscopy (AFM) imaging and fluorescence techniques, we investigate if the clustering of mApoE on gold nanoparticles (AuNP) surface may affect its performance in controlling Aß aggregation/disaggregation processes. The results showed that the ability of free mApoE to destroy preformed Aß fibrils or to hinder the Aß aggregation process is preserved after its clustering on AuNP. This allows the possibility to design multifunctional drug delivery systems with clustering of anti-amyloidogenic molecules on any NP surface without affecting their performance in controlling Aß aggregation processes.

The Extent of Human Apolipoprotein A-I Lipidation Strongly Affects the ß-Amyloid Efflux Across the Blood-Brain Barrier in vitro.

Much evidence suggests a protective role of high-density lipoprotein (HDL) and its major apolipoprotein apoA-I, in Alzheimer's disease (AD). The biogenesis of nascent HDL derived from a first lipidation of apoA-I, which is synthesized by the liver and intestine but not in the brain, in a process mediated by ABCA1. The maturation of nascent HDL in mature spherical HDL is due to a subsequent lipidation step, LCAT-mediated cholesterol esterification, and the change of apoA-I conformation. Therefore, different subclasses of apoA-I-HDL simultaneously exist in the blood circulation. Here, we investigated if and how the lipidation state affects the ability of apoA-I-HDL to target and modulate the cerebral ß-amyloid (Aß) content from the periphery, that is thus far unclear. In particular, different subclasses of HDL, each with different apoA-I lipidation state, were purified from human plasma and their ability to cross the blood-brain barrier (BBB), to interact with Aß aggregates, and to affect Aß efflux across the BBB was assessed in vitro using a transwell system. The results showed that discoidal HDL displayed a superior capability to promote Aß efflux in vitro (9 × 10-5 cm/min), when compared to apoA-I in other lipidation states. In particular, no effect on Aß efflux was detected when apoA-I was in mature spherical HDL, suggesting that apoA-I conformation, and lipidation could play a role in Aß clearance from the brain. Finally, when apoA-I folded its structure in discoidal HDL, rather than in spherical ones, it was able to cross the BBB in vitro and strongly destabilize the conformation of Aß fibrils by decreasing the order of the fibril structure (-24%) and the ß-sheet content (-14%). These data suggest that the extent of apoA-I lipidation, and consequently its conformation, may represent crucial features that could exert their protective role in AD pathogenesis.

Synthesis and Spectroscopic Characterization of 2-(het)Aryl Perimidine Derivatives with Enhanced Fluorescence Quantum Yields.

Perimidines are a particularly versatile family of heterocyclic compounds, whose properties are exploited in several applications ranging from industrial to medicinal chemistry. The molecular structure of perimidine incorporates a well-known efficient fluorophore, i.e.: 1,8-diaminonaphthalene. The high fluorescence quantum yield shared by most naphthalene derivatives, has enabled their use as stains for bio-imaging and biophysical characterizations. However, fluorescence is dramatically depressed in perimidine as well as in the few of its derivatives analysed so far to this respect. The use of perimidine-like molecules in life sciences might be notably fostered by enhancement of their fluorescence emission. Even more excitingly, the concomitance of both biologically active moieties and a fluorophore in the same molecular structure virtually discloses application of perimidines as drug compounds in state-of-art theranostics protocols. However, somewhat surprisingly, relatively few attempts were made until now in the direction of increasing the performances of perimidines as fluorescent dyes. In this work we present the synthesis and spectroscopic characterization of four perimidine derivatives designed to this aim, two of which result to be endowed with fluorescence quantum yields comparable to 1,8-diaminonaphthalene. A rationalization for such improved behaviour has been attempted employing TD-DFT calculations, which have unravelled the interrelations among bond structure, lone pair conjugation, local electron density changes and fluorescence quantum yield.

Solubilization of the chlorin TPCS2a in the presence of Pluronic® F127/Tween 80 mixtures.

The efficacy of surfactant mixtures of Pluronic® F127 and Tween 80 at overall concentration in the micromolar range and molar ratio 1:1, 1:10, and 10:1 in inhibiting aggregation of the photosensitizer meso-tetraphenyl chlorin disulphonate (TPCS2a) was investigated in aqueous media at pH 2.9 by means of steady-state absorption and fluorescence emission spectroscopy as well as time-resolved fluorescence analysis. Corresponding experiments were performed at pH 7.4 in the absence of surfactants to determine the spectroscopic properties of a monomeric sample. Aggregation resulted in a red shift of the Soret absorption band and in substantial fluorescence quenching. The fluorescence lifetime of TPCS2a was a particularly sensitive indicator of the aggregation state, as the monomer at pH 7.4 decayed with a ∼ 10 ns time constant, while aggregation resulted in subnanosecond decay. The critical micelle concentration (CMC) of the surfactant mixtures was determined spectrophotometrically in the presence of TPCS2a. The ability of the surfactant mixtures to prevent aggregation at acidic pH was evaluated at overall surfactant concentration below and above CMC. Solubilization of TPCS2a in Pluronic® F127/Tween 80 mixtures prevented aggregation of the photosensitizer at overall surfactant concentrations much lower than those needed for both pure Pluronic® F127 and pure Tween 80.

Time-Resolved Photoluminescence Microscopy Combined with X-ray Analyses and Raman Spectroscopy Sheds Light on the Imperfect Synthesis of Historical Cadmium Pigments.

Recent studies have shown that modern pigments produced after the Second Industrial Revolution are complex systems characterized by a high level of heterogeneities. Therefore, it is fundamental to adopt a multianalytical approach and highly sensitive methods to characterize the impurities present within pigments. In this work we propose time-resolved and spectrally resolved photoluminescence (PL) microscopy for the mapping of luminescent crystal defects and impurities in historical cadmium-based pigments. PL analysis is complemented by X-ray diffraction, X-ray fluorescence and Raman spectroscopies, and by scanning electron microscopy to determine the chemical composition and crystal structure of samples. The study highlights the heterogeneous and complex nature of historical samples that can be associated with the imperfect manufacturing processes tested during the period between the 1850s and 1950s. The results also allow us to speculate on a range of synthesis processes. Since it is recognized that the stability of paints can be related to pigments synthesis, this research paves the way to a wider study on the relationship between synthesis methods and deterioration of cadmium pigments and paints. This rapid and immediate approach using PL can be applied to other semiconductor pigments and real case studies.

Functionalized liposomes and phytosomes loading Annona muricata L. aqueous extract: Potential nanoshuttles for brain-delivery of phenolic compounds.

BACKGROUND: Multi-target drugs have gained significant recognition for the treatment of multifactorial diseases such as depression. Under a screening study of multi-potent medicinal plants with claimed antidepressant-like activity, the phenolic-rich Annona muricata aqueous extract (AE) emerged as a moderate monoamine oxidase A (hMAO-A) inhibitor and a strong hydrogen peroxide (H2O2) scavenger. PURPOSE: In order to protect this extract from gastrointestinal biotransformation and to improve its permeability across the blood-brain barrier (BBB), four phospholipid nanoformulations of liposomes and phytosomes functionalized with a peptide ligand promoting BBB crossing were produced. METHODS: AE and nanoformulations were characterized by HPLC-DAD-ESI-MSn, HPLC-DAD, spectrophotometric, fluorescence and dynamic light scattering methods. Cytotoxicity and permeability studies were carried out using an in vitro transwell model of the BBB, composed of immortalized human microvascular endothelial cells (hCMEC/D3), and in vitro hMAO-A inhibition and H2O2 scavenging activities were performed with all samples. RESULTS: The encapsulation/binding of AE was more efficient with phytosomes, while liposomes were more stable, displaying a slower extract release over time. In general, phytosomes were less toxic than liposomes in hCMEC/D3 cells and, when present, cholesterol improved the permeability across the cell monolayer of all tested nanoformulations. All nanoformulations conserved the antioxidant potential of AE, while phosphatidylcholine interfered with MAO-A inhibition assay. CONCLUSIONS: Overall, phytosome formulations registered the best performance in terms of binding efficiency, enzyme inhibition and scavenging activity, thus representing a promising multipotent phenolic-rich nanoshuttle for future in vivo depression treatment.

Excited state dynamics of bis-dehydroxycurcumin tert-butyl ester, a diketo-shifted derivative of the photosensitizer curcumin.

Bis-dehydroxycurcumin tert-butyl ester (K2T23) is a derivative of the natural spice curcumin. Curcumin is widely studied for its multiple therapeutic properties, including photosensitized cytotoxicity. However, the full exploitation of curcumin phototoxic potential is hindered by the extreme instability of its excited state, caused by very efficient non radiative decay by means of transfer of the enolic proton to the nearby keto oxygen. K2T23 is designed to exhibit a tautomeric equilibrium shifted toward the diketo conformers with respect to natural curcumin. This property should endow K2T23 with superior excited-state stability when excited in the UVB band, i.e., in correspondence of the diketo conformers absorption peaks, making this compound an interesting candidate for topical photodynamic therapy of, e.g., skin tumors or oral infections. In this work, the tautomeric equilibrium of K2T23 between the keto-enolic and diketo conformers is assessed in the ground state in several organic solvents by UV-visible absorption and by nuclear magnetic resonance. The same tautomeric equilibrium is also probed in the excited-state in the same environments by means of steady-state fluorescence and time-correlated single-photon counting measurements. These techniques are also exploited to elucidate the excited state dynamics and excited-state deactivation pathways of K2T23, which are compared to those determined for several other curcuminoids characterized in previous works of ours. The ability of K2T23 in photosensitizing the production of singlet oxygen is compared with that of curcumin.

Time-resolved homo-FRET studies of biotin-streptavidin complexes.

Förster resonance energy transfer is a mechanism of fluorescence quenching that is notably useful for characterizing properties of biomolecules and/or their interactions. Here we study water-solutions of Biotin-Streptavidin complexes, in which Biotin is labeled with a rigidly-bound fluorophore that can interact by Förster resonance energy transfer with the fluorophores labeling the other, up to three, Biotins of the same complex. The fluorophore, Atto550, is a Rhodamine analogue. We detect the time-resolved fluorescence decay of the fluorophores with an apparatus endowed with single-photon sensitivity and temporal resolution of ~30ps. The decay profiles we observe for samples containing constant Biotin-Atto550 conjugates and varying Streptavidin concentrations are multi-exponential. Each decay component can be associated with the rate of quenching exerted on each donor by each of the acceptors that label the other Biotin molecules, depending on the binding site they occupy. The main features that lead to this result are that (i) the transition dipole moments of the up-to-four Atto550 fluorophores that label the complexes are fixed as to both relative positions and mutual orientations; (ii) the fluorophores are identical and the role of donor in each Biotin-Streptavidin complex is randomly attributed to the one that has absorbed the excitation light (homo-FRET). Obviously the high-temporal resolution of the excitation-detection apparatus is necessary to discriminate among the fluorescence decay components.

Platinum-Based Drugs and DNA Interactions Studied by Single-Molecule and Bulk Measurements.

Platinum-containing molecules are widely used as anticancer drugs. These molecules exert cytotoxic effects by binding to DNA through various mechanisms. The binding between DNA and platinum-based drugs hinders the opening of DNA, and therefore, DNA duplication and transcription are severely hampered. Overall, impeding the above-mentioned important DNA mechanisms results in irreversible DNA damage and the induction of apoptosis. Several molecules, including multinuclear platinum compounds, belong to the family of platinum drugs, and there is a body of research devoted to developing more efficient and less toxic versions of these compounds. In this study, we combined different biophysical methods, including single-molecule assays (magnetic tweezers) and bulk experiments (ultraviolet absorption for thermal denaturation) to analyze the differential stability of double-stranded DNA in complex with either cisplatin or multinuclear platinum agents. Specifically, we analyzed how the binding of BBR3005 and BBR3464, two representative multinuclear platinum-based compounds, to DNA affects its stability as compared with cisplatin binding. Our results suggest that single-molecule approaches can provide insights into the drug-DNA interactions that underlie drug potency and provide information that is complementary to that generated from bulk analysis; thus, single-molecule approaches have the potential to facilitate the selection and design of optimized drug compounds. In particular, relevant differences in DNA stability at the single-molecule level are demonstrated by analyzing nanomechanically induced DNA denaturation. On the basis of the comparison between the single-molecule and bulk analyses, we suggest that transplatinated drugs are able to locally destabilize small portions of the DNA chain, whereas other regions are stabilized.

Novel Antitransferrin Receptor Antibodies Improve the Blood-Brain Barrier Crossing Efficacy of Immunoliposomes.

Surface functionalization with antitransferrin receptor (TfR) mAbs has been suggested as the strategy to enhance the transfer of nanoparticles (NPs) across the blood-brain barrier (BBB) and to carry nonpermeant drugs from the blood into the brain. However, the efficiency of BBB crossing is currently too poor to be used in vivo. In the present investigation, we compared 6 different murine mAbs specific for different epitopes of the human TfR to identify the best performing one for the functionalization of NPs. For this purpose, we compared the ability of mAbs to cross an in vitro BBB model made of human brain capillary endothelial cells (hCMEC/D3). Liposomes functionalized with the best performing mAb (MYBE/4C1) were uptaken, crossed the BBB in vitro, and facilitated the BBB in vitro passage of doxorubicin, an anticancer drug, 3.9 folds more than liposomes functionalized with a nonspecific IgG, as assessed by confocal microscopy, radiochemical techniques, and fluorescence, and did not modify the cell monolayer structural or functional properties. These results show that MYBE/4C1 antihuman TfR mAb is a powerful resource for the enhancement of BBB crossing of NPs and is therefore potentially useful in the treatment of neurologic diseases and disorders including brain carcinomas.

Fluorimetric detection of the earliest events in amyloid ß oligomerization and its inhibition by pharmacologically active liposomes.

BACKGROUND: Amyloid ß (Aß) peptide aggregation is the main molecular mechanism underlying the development of Alzheimer's disease, the most widespread form of senile dementia worldwide. Increasing evidence suggests that the key factor leading to impaired neuronal function is accumulation of water-soluble Aß oligomers rather than formation of the senile plaques created by the deposition of large fibrillary aggregates of Aß. However, several questions remain about the preliminary steps and the progression of Aß oligomerization. METHODS: We show that the initial stages of the aggregation of fluorescently labeled Aß can be determined with a high degree of precision and at physiological (i.e., nanomolar) concentrations by using either steady-state fluorimetry or time-correlated single-photon counting. RESULTS: We study the dependence of the oligomerization extent and rate on the Aß concentration. We determine the chemical binding affinity of fluorescently labeled Aß for liposomes that have been recently shown to be pharmacologically active in vivo, reducing the Aß burden within the brain. We also probe their capacity to hinder the Aß oligomerization process in vitro. CONCLUSIONS: We introduced a fluorescence assay allowing investigation of the earliest steps of Aß oligomerization, the peptide involved in Alzheimer's disease. The assay proved to be sensitive even at Aß concentrations as low as those physiologically observed in the cerebrospinal fluid. GENERAL SIGNIFICANCE: This work represents an extensive and quantitative study on the initial events of Aß oligomerization at physiological concentration. It may enhance our comprehension of the molecular mechanisms leading to Alzheimer's disease, thus paving the way to novel therapeutic strategies.