Development of a Nanoparticle-Based Approach for the Blood-Brain Barrier Passage in a Murine Model of Amyotrophic Lateral Sclerosis.

The development of nanoparticles (NPs) to enable the passage of drugs across blood-brain barrier (BBB) represents one of the main challenges in neuropharmacology. In recent years, NPs that are able to transport drugs and interact with brain endothelial cells have been tested. Here, we investigated whether the functionalization of avidin-nucleic-acid-nanoassembly (ANANAS) with apolipoprotein E (ApoE) would allow BBB passage in the SOD1G93A mouse model of amyotrophic lateral sclerosis. Our results demonstrated that ANANAS was able to transiently cross BBB to reach the central nervous system (CNS), and ApoE did not enhance this property. Next, we investigated if ANANAS could improve CNS drug delivery. To this aim, the steroid dexamethasone was covalently linked to ANANAS through an acid-reversible hydrazone bond. Our data showed that the steroid levels in CNS tissues of SOD1G93A mice treated with nanoformulation were below the detection limit. This result demonstrates that the passage of BBB is not sufficient to guarantee the release of the cargo in CNS and that a different strategy for drug tethering should be devised. The present study furthermore highlights that NPs can be useful in improving the passage through biological barriers but may limit the interaction of the therapeutic compound with the specific target.

The mode of dexamethasone decoration influences avidin-nucleic-acid-nano-assembly organ biodistribution and in vivo drug persistence.

Avidin-Nucleic-Acid-NanoASsemblies (ANANAS) possess natural tropism for the liver and, when loaded with dexamethasone, reduce clinical progression in an autoimmune hepatitis murine model. Here, we investigated the linker chemistry (hydrazide-hydrazone, Hz-Hz, or carbamate hydrazide-hydrazone, Cb-Hz bond) and length (long, 5 kDa PEG, or short, 5-6 carbons) in biotin-dexamethasone conjugates used for nanoparticle decoration through in vitro and in vivo studies. All four newly synthesized conjugates released the drug at acidic pH only. In vitro, the Hz-Hz and the PEG derivatives were less stable than the Cb-Hz and the short chain ones, respectively. Once injected in healthy mice, dexamethasone location in the PEGylated ANANAS outer layer favors liver penetration and resident macrophages uptake, while drug Hz-Hz, but not Cb-Hz, short spacing prolongs drug availability. In conclusion, the tight modulation of ANANAS decoration can significantly influence the host interaction, paving the way for the development of steroid nanoformulations suitable for different pharmacokinetic profiles.

Dexamethasone Conjugation to Biodegradable Avidin-Nucleic-Acid-Nano-Assemblies Promotes Selective Liver Targeting and Improves Therapeutic Efficacy in an Autoimmune Hepatitis Murine Model.

Steroids are the standard therapy for autoimmune hepatitis (AIH) but the long-lasting administration is hampered by severe side effects. Methods to improve the tropism of the drug toward the liver are therefore required. Among them, conjugation to nanoparticles represents one possible strategy. In this study, we exploited the natural liver tropism of Avidin-Nucleic-Acid-Nano-Assemblies (ANANAS) to carry dexamethasone selectively to the liver in an AIH animal model. An acid-labile biotin-hydrazone linker was developed for reversible dexamethasone loading onto ANANAS. The biodistribution, pharmacokinetics and efficacy of free and ANANAS-linked dexamethasone (ANANAS-Hz-Dex) in healthy and AIH mice were investigated upon intraperitoneal administration. In ANANAS-treated animals, the free drug was detected only in the liver. Super-resolution microscopy showed that nanoparticles segregate inside lysosomes of liver immunocompetent cells, mainly involved in AIH progression. In agreement with these observational results, chronic low-dose treatment with ANANAS-Hz-Dex reduced the expression of liver inflammation markers and, in contrast to the free drug, also the levels of circulating AIH-specific autoantibodies. These data suggest that the ANANAS carrier attenuates AIH-related liver damage without drug accumulation in off-site tissues. The safety and biodegradability of the ANANAS carrier make this formulation a promising tool for the treatment of autoimmune liver disorders.

Improvement and extension of anti-EGFR targeting in breast cancer therapy by integration with the Avidin-Nucleic-Acid-Nano-Assemblies.

Nowadays, personalized cancer therapy relies on small molecules, monoclonal antibodies, or antibody-drug conjugates (ADC). Many nanoparticle (NP)-based drug delivery systems are also actively investigated, but their advantage over ADCs has not been demonstrated yet. Here, using the Avidin-Nucleic-Acid-Nano-Assemblies (ANANAS), a class of polyavidins multifuctionalizable with stoichiometric control, we compare quantitatively anti-EGFR antibody(cetuximab)-targeted NPs to the corresponding ADC. We show that ANANAS tethering of cetuximab promotes a more efficient EGFR-dependent vesicle-mediated internalization. Cetuximab-guided ANANAS carrying doxorubicin are more cytotoxic in vitro and much more potent in vivo than the corresponding ADC, leading to 43% tumor reduction at low drug dosage (0.56 mg/kg). Advantage of cetuximab-guided ANANAS with respect to the ADC goes beyond the increase in drug-to-antibody ratio. Even if further studies are needed, we propose that NP tethering could expand application of the anti-EGFR antibody to a wider number of cancer patients including the KRAS-mutated ones, currently suffering from poor prognosis.

A New ELISA Using the ANANAS Technology Showing High Sensitivity to diagnose the Bovine Rhinotracheitis from Individual Sera to Pooled Milk.

Diagnostic tests for veterinary surveillance programs should be efficient, easy to use and, possibly, economical. In this context, classic Enzyme linked ImmunoSorbent Assay (ELISA) remains the most common analytical platform employed for serological analyses. The analysis of pooled samples instead of individual ones is a common procedure that permits to certify, with one single test, entire herds as "disease-free". However, diagnostic tests for pooled samples need to be particularly sensitive, especially when the levels of disease markers are low, as in the case of anti-BoHV1 antibodies in milk as markers of Infectious Bovine Rhinotracheitis (IBR) disease. The avidin-nucleic-acid-nanoassembly (ANANAS) is a novel kind of signal amplification platform for immunodiagnostics based on colloidal poly-avidin nanoparticles that, using model analytes, was shown to strongly increase ELISA test performance as compared to monomeric avidin. Here, for the first time, we applied the ANANAS reagent integration in a real diagnostic context. The monoclonal 1G10 anti-bovine IgG1 antibody was biotinylated and integrated with the ANANAS reagents for indirect IBR diagnosis from pooled milk mimicking tank samples from herds with IBR prevalence between 1 to 8%. The sensitivity and specificity of the ANANAS integrated method was compared to that of a classic test based on the same 1G10 antibody directly linked to horseradish peroxidase, and a commercial IDEXX kit recently introduced in the market. ANANAS integration increased by 5-fold the sensitivity of the 1G10 mAb-based conventional ELISA without loosing specificity. When compared to the commercial kit, the 1G10-ANANAS integrated method was capable to detect the presence of anti-BHV1 antibodies from bulk milk of gE antibody positive animals with 2-fold higher sensitivity and similar specificity. The results demonstrate the potentials of this new amplification technology, which permits improving current classic ELISA sensitivity limits without the need for new hardware investments.

Detection of a fluorescent-labeled avidin-nucleic acid nanoassembly by confocal laser endomicroscopy in the microvasculature of chronically inflamed intestinal mucosa.

Inflammatory bowel diseases are chronic gastrointestinal pathologies causing great discomfort in both children and adults. The pathogenesis of inflammatory bowel diseases is not yet fully understood and their diagnosis and treatment are often challenging. Nanoparticle-based strategies have been tested in local drug delivery to the inflamed colon. Here, we have investigated the use of the novel avidin-nucleic acid nanoassembly (ANANAS) platform as a potential diagnostic carrier in an experimental model of inflammatory bowel diseases. Fluorescent- labeled ANANAS nanoparticles were administered to mice with chemically induced chronic inflammation of the large intestine. Localization of mucosal nanoparticles was assessed in vivo by dual-band confocal laser endomicroscopy. This technique enables characterization of the mucosal microvasculature and crypt architecture at subcellular resolution. Intravascular nanoparticle distribution was observed in the inflamed mucosa but not in healthy controls, demonstrating the utility of the combination of ANANAS and confocal laser endomicroscopy for highlighting intestinal inflammatory conditions. The specific localization of ANANAS in inflamed tissues supports the potential of this platform as a targeted carrier for bioactive moieties in the treatment of inflammatory bowel disease.

Molecular mechanisms of antiproliferative effects induced by Schisandra-derived dibenzocyclooctadiene lignans (+)-deoxyschisandrin and (-)-gomisin N in human tumour cell lines.

A different behavior of the two dibenzocyclooctadiene lignans (+)-deoxyschisandrin (1) and (-)-gomisin N (2), from Schisandra chinensis fruits, was observed against two human tumour cell lines, (2008 and LoVo). These lignans inhibited cell growth in a dose-dependent manner on both cell lines, but inducing different types of cell death. In particular, (+)-deoxyschisandrin (1) caused apoptosis in colon adenocarcinoma cells (LoVo) but not in ovarian adenocarcinoma cells (2008), while (-)-gomisin N (2) induced apoptosis on both the cell lines used. Mitochondrial-mediated pathway was not involved in apoptotic stimuli. Both compounds caused G2/M phase cell growth arrest correlated with tubulin polymerization.

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.

C2362F mutation gives rise to an ADAMTS13-resistant von Willebrand factor.

von Willebrand factor (VWF) multimers result from proteolysis by the metalloprotease ADAMTS13. Since C2362F-VWF features abnormally large multimers with their triplet oligomer structure replaced by a diffuse smear, we explored the susceptibility of C2362F-VWF to ADAMTS13. VWF-enriched blood samples, obtained by cryoethanol precipitation of plasma from a patient with von Willebrand disease (VWD) homozygous for the C2362F mutation and a normal subject, were submitted to cleavage by recombinant ADAMTS13 under static conditions in the presence of urea. C2362F-VWF proved completely ADAMTS13-resistant in vitro. At any concentration of recombinant ADAMTS13 (from 0.1 µM to 1 µM), there was no evidence of the abnormally large VWF multimers of C2362F-VWF disappearing, nor any increased representation of triplet multimer bands, unlike the situation seen in normal VWF. This is due partly to a defective ADAMTS13 binding to C2362F-VWF under static conditions, as seen in both the patient's and recombinant mutated VWF proteins. These findings were associated with a significantly shorter than normal survival of C2362F-VWF after DDAVP, demonstrating that proteolysis and VWF survival may be independent phenomena. Our findings clearly demonstrate that the loss of cysteine 2362 makes VWF resistant to proteolysis by ADAMTS13, at least partly due to an impaired ADAMTS13 binding to VWF. This suggests that the B2 domain of VWF is involved in modulating ADAMTS13 binding to VWF and the consequent proteolytic process. The C2362F-VWF mutation also enables a new abnormality to be identified in the VWF-ADAMTS13 relationship, i.e. an ADAMTS13-resistant VWF.

Synthesis and chromatography-free purification of PNA-PEO conjugates for the functionalisation of gold sensors.

Peptide Nucleic Acids (PNAs) linked to high molecular weight (MW) poly(ethylene oxide) (PEO) derivatives could be useful conjugates for the direct functionalisation of gold surfaces dedicated to Surface Plasmon Resonance (SPR)-based DNA sensing. However their use is hampered by the difficulty to obtain them through a convenient and economical route. In this work we compared three synthetic strategies to obtain PNA-high MW PEO conjugates composed of (a) a 15-mer PNA sequence as the probe complementary to genomic DNA of ]Mycobacterium tuberculosis, (b) a PEO moiety (2 or 5 KDa MW) and (c) a terminal trityl-protected thiol necessary (after acidic deprotection) for grafting to gold surfaces. The 15-mer PNA was obtained by solid-phase synthesis. Its amino terminal group was later condensed to bi-functional PEO derivatives (2 and 5 KDa MW) carrying a Trt-cysteine at one end and a carboxyl group at the other end. The reaction was carried out either in solution, using HATU or PyOxim as coupling agents, or through the solid-phase approach, with 49.6%, 100% and 5.2% yield, respectively. A differential solvent extraction strategy for product purification without the need for chromatography is described. The ability of the 5 KDa PEO conjugate to function as a probe for complementary DNA detection was demonstrated using a Grating-Coupling Surface Plasmon Resonance (GC-SPR) system. The optimized PEO conjugation and purification protocols are economical and simple enough to be reproduced also within laboratories that are not highly equipped for chemical synthesis.

Characterization of multifunctional nanosystems based on the avidin-nucleic acid interaction as signal enhancers in immuno-detection.

The Avidin-Nucleic-Acids-Nano-Assembly (ANANAS) is a kind of soft poly avidin nanoparticle originating from the high affinity interaction between avidin and the nucleic acids. In this work we investigated the possibility of transforming ANANAS cores into stoichiometrically controlled multifunctional nanoparticles through a "one-pot" procedure, and we measured in a quantitative way their ability to work as reagents for enhanced immunodiagnostic detection. Initially, we measured the ANANAS loading capability for biotinylated proteins of different nature. About 200 molecules of biotin-horseradish-peroxidase (40KDa b-HRP) and 60 molecules of biotin-immunoglobulin-G (150KDa b-IgG) could be accommodated onto each nanoparticle, showing that steric limitations dictate the number of loadable entities. Stoichiometrically controlled functional assemblies were generated by mixing core particles with subsaturating amounts of b-HRP and b-IgG. When applied as detection reagents in an Enzyme-Linked-ImmunoSorbed-Assay (ELISA), these assemblies were up to two-orders of magnitude more sensitive than commercial HRP-based reagents. Assemblies of different composition displayed different efficacy, indicating that the system functionality can be fine-tuned. Within-assay variability (CV%), measured to assess if the assembly procedure is reproducible, was within 10%. Stability experiments demonstrated that the functionalyzed assemblies are stable in solution for more than one week. In principle, any biotinylated function can be loaded onto the core particle, whose high loading capacity and tunability may open the way toward further application in biomedicine.

Evaluation of cytotoxic activity of Schisandra chinensis lignans.

Using exhaustive chromatographic separation we have isolated (-)-tigloyl-deangeloyl-gomisin F as a novel dibenzocyclooctadiene lignan from schisandra chinensis. With the help of HPLC, we further isolated (+)-schisandrin, (+)-deoxyschisandrin, (+)-γ-schisandrin, (-)-gomisin J, (+)-gomisin A, (-)-gomisin N, (-)-tigloyl-gomisin P, (-)-wuweizisu C, (-)-gomisin D, rubrisandrin A, (-)-gomisin G, (+)-gomisin K (3) and (-)-schisantherin C. A full NMR description of (-)-schisantherin C was carried out with the aim to confirm previous reports of its structure. Compounds isolated were identified on the basis of UV, IR, (1)H- and (13)C-NMR and MS. The cytotoxicity of lignans was tested for the BY-2 cell line alone and as a synergistic effect with the cytotoxic agent camptothecin. Lignans showed various toxicity and synergistic and antagonistic effects on camptothecin-induced cytotoxicity. Cytotoxicity against colon cancer cell line LoVo was also tested.